THE REGULATORY ROLES OF TRAF4 IN IL-17 RECEPTOR
MEDIATED SIGNALING AND DISEASE PATHOLOGY
BY
JAROD A. ZEPP
Submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy
Dissertation Advisor:
Dr. Xiaoxia Li
Department of Molecular Medicine
CASE WESTERN RESERVE UNIVERSITY
August 2014
1 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Jarod A. Zepp candidate for the degree of DOCTOR OF PHILOSPHY
(signed)
Committee Chair
Timothy Nilsen, Ph.D.
Committee Member, Advisor
Xiaoxia Li, Ph.D.
Committee Member
Serpil Erzurum, M.D.
Committee Member
Paul Fox, Ph.D.
Committee Member
Olga Stenina-Adognravi, Ph.D.
Date of Defense
May 30, 2014
*We also certify that written approval has been obtained
for any proprietary material contained therein.
2
Copyright © 2014. Jarod A. Zepp Copyright © 2012. The American Association of Immunologists, Inc. All rights reserved
3
Dedication
To my courageous, compassionate and devoted mother.
To my grandparents Arthur & Ann Thompson.
To my grandfather Robert Zepp.
4 Table of Contents: ♦ Title page...... 1 ♦ Committee signature sheet...... 2 ♦ Dedication page...... 3 ♦ Table of Contents...... 4 ♦ List of Figures...... 7 ♦ Acknowledgements...... 9
♦ Abstract
♦ Chapter 1: Introduction to the Interleukin-17 cytokine family...... 13 I. Interleukin-17 cytokine family...... 14 II. ACT1; A critical adaptor protein for IL-17R signaling...... 16 a. Discovery of ACT1...... 16 b. The STIR domain superfamily...... 18 c. ACT1 is a U-box type E3 ubiquitin ligase...... 19 d. ACT1, the double-edged sword...... 22 III. IL-17 and IL-25-associated disease pathology...... 26 a. Pathogenic Th17 cells and Autoimmune Diseases...... 26 b. Th17 and IL-17 in EAE, a murine model for the immunopathogenesis of multiple sclerosis...... 27 c. IL-17-induced ACT1-mediated signaling in CNS resident cells is necessary for Th17-induced CNS inflammation and EAE...... 32 d. IL-17 and IL-25 mediate allergic airway inflammation and airway hyperresponsiveness...... 34 IV. IL-17 and IL-25 signaling...... 38 a. TNF-receptor associated factor (TRAF) protein family...... 38 b. IL-17 and IL-25 signaling, a current overview...... 41
♦ Chapter 2: TNF Receptor-associated factor 4 restricts IL-17-mediated pathology and signaling processes...... 44 I. Abstract...... 45 II. Introduction...... 46 III. Materials and Methods...... 48 IV. Results a. IL-17–induced signaling and gene expression are increased in TRAF4- deficient cells...... 52 b. TRAF4 deficiency exacerbates EAE severity...... 56 c. ACT1-TRAF4 complex is distinct from other ACT1-TRAF complexes...... 60 d. TRAF4 competes with TRAF6 for ACT1 TRAF binding sites...... 62 V. Discussion...... 68
5
♦ Chapter 3: TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation...... 71 I. Abstract...... 72 II. Introduction...... 73 III. Materials and Methods...... 76 IV. Results a. Differential requirements for TRAF proteins in the IL-25 response.83 b. TRAF4 is required for IL-25-dependent airway inflammation...... 86 c. TRAF4 is required for IL-25 responses in T-cells and airway epithelial cells...... 9 4 d. TRAF4 and SMURF2 cooperate to mediate the IL-25 response..... 102 e. TRAF4 and SMURF2 are required for IL-25-induced degradation of the inhibitory molecule DAZAP2...... 108 f. IL-25R recruits DAZAP2 through p-Tyr 355 residing within the SEFIR domain...... 117 g. IL-25R Y355F mutant mediates IL-25 response in the absence of TRAF4...... 122 V. Discussion...... 124
♦ Chapter 4: Conclusions and future study...... 129 I. Introduction...... 130 II. TRAF4 in the IL-17 signaling pathway...... 131 III. TRAF4/SMURF2 in the IL-25 pathway...... 132 IV. Future study...... 134 a. TRAF4 as a therapeutic target in inflammatory diseases...... 134 b. IL-25R-dependent STAT activation...... 135 c. IL-25R as a therapeutic target and biomarker in respiratory Disorders...... 137 V. Final Remarks...... 138
♦ Appendix 1...... 140 ♦ Appendix 2...... 141 ♦ Appendix 3...... 142 ♦ Bibliography...... 144
6 List of Figures
Figure 1-1 IL-17 cytokine and receptor family...... 15
Figure 1-2 Act1 structure and IL-17 signaling cascade...... 18
Figure 1-3 Th17 cells in the Initiation and effector stages of EAE...... 31
Figure 1-4 IL-17 and IL-25 in airway inflammation...... 37
Figure 1-5 TNF-receptor Associated Factor (TRAF) Family...... 40
Figure 1-6 IL-17 and IL-25 signaling...... 43
Figure 2-1 A-B, IL-17 induced signaling and gene expression in
TRAF4-deficient primary cells...... 54
Figure 2-1 C-E, IL-17 induced signaling and gene expression in
TRAF4-deficient primary cells...... 55
Figure 2-2 A, TRAF4 deficiency exacerbates Th17 mediated EAE...... 57
Figure 2-2 B, TRAF4 deficiency exacerbates Th17 mediated EAE...... 58
Figure 2-2 C-E, TRAF4 deficiency exacerbates Th17 mediated EAE.. 59
Figure 2-3 Distinct Act1-TRAF interactions form following IL-17
stimulation...... 61
Figure 2-4 A-B, TRAF4 restricts Act1/TRAF6 interaction...... 64
Figure 2-4 C-D, TRAF4 restricts Act1/TRAF6 interaction...... 65
Figure 2-4 E, TRAF4 restricts Act1/TRAF6 interaction...... 66
Figure 2-5 Working model of IL-17 signaling cascade...... 67
Figure 3-1 Screening for TRAF involvement in the
IL-25R-dependent response...... 85
7 Figure 3-2 A-B, TRAF4 regulates IL-25 responses in vivo...... 87
Figure 3-2 C, TRAF4 regulates IL-25 responses in vivo...... 88
Figure 3-2 D-E, TRAF4 regulates IL-25 responses in vivo...... 89
Figure 3-3 Enhanced IL-17A-induced airway inflammation in
TRAF4-deficient mice...... 90
Figure 3-4 A, TRAF4 mediates allergic airway inflammation...... 92
Figure 3-4 B, TRAF4 mediates allergic airway inflammation...... 93
Figure 3-4 C, TRAF4 mediates allergic airway inflammation...... 94
Figure 3-5 A-B, Cell-intrinsic IL-25 responses are TRAF4-dependent....96
Figure 3-5 C-D, Cell-intrinsic IL-25 responses are TRAF4-dependent... 97
Figure 3-5 E, Cell-intrinsic IL-25 responses are TRAF4-dependent...... 98
Figure 3-5 F-G Cell-intrinsic IL-25 responses are TRAF4-dependent...100
Figure 3-5 H, Cell-intrinsic IL-25 responses are TRAF4-dependent..... 101
Figure 3-6 A-D, SMURF2 is a positive mediator of the
IL-25-response...... 101
Figure 3-6 E-G, SMURF2 is a positive mediator of the
IL-25-response...... 106
Figure 3-6 H-I, SMURF2 is a positive mediator of the
IL-25-response...... 107
Figure 3-7 A-D, DAZAP2 negatively impacts IL-25 responses...... 109
Figure 3-7 E-G, DAZAP2 negatively impacts IL-25 responses...... 111
Figure 3-7 H, DAZAP2 negatively impacts IL-25 responses...... 112
8 Figure 3-8 A, IL-25 stimulation promotes DAZAP2 degradation...... 113
Figure 3-8 B, IL-25 stimulation promotes DAZAP2 degradation...... 114
Figure 3-8 C, IL-25 stimulation promotes DAZAP2 degradation...... 115
Figure 3-8 D-E, IL-25 stimulation promotes DAZAP2 degradation....116
Figure 3-9 A, IL-25R contains tyrosine residues that modulate
its function...... 118
Figure 3-9 B-D, IL-25R contains tyrosine residues that modulate
its function...... 119
Figure 3-9 E-H, IL-25R contains tyrosine residues that modulate
its function...... 121
Figure 3-9 I, IL-25R contains tyrosine residues that modulate
its function...... 123
Figure 4-1 TRAF4 is required for P-STAT5 activation...... 136
9 Acknowledgements
The work presented in this dissertation was accomplished through the assistance and support of many highly skilled and dedicated scientists and clinicians. I am indebted and deeply grateful for their contributions. I would like to acknowledge my research mentor Dr. Xiaoxia Li. This work would not be possible without Xiaoxia’s guidance, advice, patience and unconditional support. I am grateful for Xiaoxia’s passion for science and hypothesis-driven research. Xiaoxia’s constant desires to pursue and test new biological concepts are traits that I hope to maintain myself throughout my own scientific career. In addition I would also like to thank the incredible scientists that Xiaoxia has employed in her lab. They were my greatest colleagues and I thank you, Fatih Gulen, Junjie Zhao, Ling Wu, Caini Liu, Zizhen Kang, Brad Martin, Hao Zhou, Wen Qian, Kate Bulek, Tomek Herjan, Rachel Yu, Chenhui Wang, Chunfang Gu and Xing Chen. I am deeply honored to have the support of the distinguished scientists that served on my thesis committee. I am thankful for Dr. Nilsen’s guidance especially during the early stages of my research. I would like to thank Dr. Fox and Dr. Stenina for their insightful comments and advice. I am thankful to my clinical mentor, Dr. Erzurum for sharing her time in the clinic with me. I am also thankful for her encouragement and guidance throughout my thesis work. I would also like to thank the administrators that helped me throughout my graduate study particularly Dr. Marcia Jarrett, Erica Healey-Pavlik, Jan Kodish, Gail Lannum and Lisa Franklin.
10
The Regulatory Roles Of Traf4 Il-17 Receptor Mediated Signaling And Disease
Pathology
Abstract
By
JAROD A. ZEPP
The IL-17 cytokine family members, IL-17 and IL-25, have both been implicated in the pathogenesis of numerous inflammatory disorders including multiple sclerosis and asthma. Although IL-17 and IL-25 belong to the same cytokine family, they induce distinct gene transcriptional programs that drive contrasting physiological responses. For instance, administration of IL-17 to the lungs of mice drives the accumulation of neutrophils whereas IL-25 mediates the recruitment of eosinophils.
We have shown that binding of IL-17 and IL-25 to their respective receptors leads to the recruitment of the adaptor protein ACT1 and subsequent activation of TNF- receptor associated factor (TRAF) molecules. Although numerous factors have been identified downstream of IL-17, the IL-25 pathway and factors that distinguish it from IL-17, remain obscure. Here, we tested the role of TRAF4 in the IL-17 and IL-
25 pathways. In the IL-17 pathway, TRAF4 interacts with the TRAF-binding domains in ACT1 and TRAF4-deficiency enhances IL-17-induced ACT1-dependent recruitment of TRAF6 and subsequent NFkB activation. Moreover, TRAF4-deficient mice are more susceptible to IL-17-driven disease pathogenesis in a mouse model of multiple sclerosis. In contrast, we find that TRAF4-deficiency abolishes the IL-25
11 response. While injection of IL-25 led to eosinophil recruitment and induction of cytokines, IL-4, IL-5 and IL-13 in the lungs of control mice, this response was diminished in TRAF4-deficient mice. We identified a unique signaling nexus composed of TRAF4 and an E3-ligase SMURF2. An IL-25-receptor specific interacting molecule, known as DAZAP2, inhibits IL-25-induced signaling, gene expression and ACT1 recruitment. Upon IL-25-stimulation TRAF4-dependent
SMURF2 recruitment was required for K48-linked ubiquitination and degradation of
DAZAP2 to subsequently facilitate IL-25R/ACT1 binding. Together these findings implicate TRAF4 as a crucial regulatory molecule in both IL-17 and IL-25 signaling pathways.
12 Chapter 1:
Introduction to the Interleukin-17 Cytokine Family
Portions of this chapter are published in;
Zepp JA & Wu L, Li X. 2011. IL-17 receptor signaling and T helper 17- mediated autoimmune demyelinating disease. Trends in Immunology. May;32(5);232-9. (permissions Appendix 1)
Wu L & Zepp JA and Li X. 2012. Function of Act1 in IL-17 family signaling and autoimmunity. Current Topics in Innate Immunity II; Advances in experimental medicine and biology. 949:223-35. (permissions Appendix 2)
13 I. Interleukin-17 cytokine family
Homology-based cloning has revealed six IL-17 family members, termed IL-
17A to IL17F. IL-17 (IL-17A), produced by Th17 cells, is the prototypic IL-17 family member, exerting its actions either as a homodimer or as a heterodimer with
IL-17F (Kawaguchi et al., 2001) (Gaffen, 2008). IL-17 coordinates local tissue inflammation through the upregulation of pro-inflammatory cytokines and chemokines such as IL-6, G-CSF, TNFa, IL-1, CXCL1(KC), CCL2(MCP-1),
CXCL2(MIP-2), CCL7(MCP-3), and CCL20(MIP-3A). In addition, it induces matrix metalloproteases (MMPs) that allow activated T cells to penetrate the extracellular matrix. Recent studies have shown that IL-17 signals through a heteromeric receptor complex formed by IL-17R (IL-17RA) and IL-17RC, which are single-pass transmembrane proteins expressed by a variety of cells including astrocytes and microglia (Fig. 1-1). IL-17RA can be bound by both IL-17A and IL-17F, but with
10-fold more affinity for IL-17A than for IL-17F. IL-17R (IL-17RA) and IL-17RC belong to the IL-17 receptor family, including three additional members IL-17RB, IL-
17RD and IL-17RE. While both IL-17E and IL-17B bind IL-17RB, inducing Th2 cytokines, the ligand for IL-17RD is still unknown. IL-17C, the ligand for IL-17RE, mediate anti-microbial defense at mucosal sites such as the in the intestine. It is important to note that recent studies have shown binding of the first IL-17 receptor subunit to the ligand modulates the affinity and specificity of the second-binding event, thereby promoting heterodimeric versus homodimeric complex formation (Ely et al., 2009).
14
Figure 1-1. IL-17 cytokine and receptor family. The IL-17 cytokine family includes IL-17 A-F, which are predicted to form homo- and heterodimeric interactions that are necessary for signaling. There are also five IL-17 receptor subunits, of which IL-17RA, -RC and –RB are the best described. The receptor subunit, IL-17RA, is common for IL-17A, IL-17C, IL-17F and IL-17E (IL-25) driven gene expression. IL-17A and IL-17F bind the receptor complex IL-17RA/IL-17RC to drive inflammatory gene expression. IL-25 binds to the IL-17RA/IL-17RB complex to mediate its effects on Th2 homeostasis. IL-17C binds to the IL- 17RA/IL-17RE complex to mediate anti-microbial peptide and chemokine expression and it also promotes Th17 T-cell effector function.
15 II. ACT1; A critical adaptor protein for IL-17R signaling
Discovery of ACT1
Under the control of various inflammatory and pathogen derived stimuli, the transcription factor NFkB is a central mediator of gene expression. Upon its activation, NFkB modulates the expression of many target genes including those for cytokines, chemokines, and cell surface receptors among many others. Under normal conditions NFκB is sequestered in the cytoplasm by IkB inhibitory proteins. Upon stimulation with various extracellular stimuli (including Toll-like receptor ligands, IL-
1 and TNFα), the IκBα protein is phosphorylated by IκBα kinase thereby releasing
NFκB and allowing its translocation to the cell nucleus. The signaling pathways that activate NFκB converge at the IKK complex that is composed of three subunits, the catalytic subunits IKKα and –β and the regulatory subunit IKKγ (Li and Verma,
2002).
Using a screening approach for novel NFκB activators, Act1/CIKS was identified. Using an NFκB-dependent selectable marker, Act1 (NFκB activator 1) was discovered due to its ability to activate NFκB (Li et al., 2000). Over-expression of
Act1 leads to constitutive activation of NFκB as well as JNK. Furthermore it was shown that Act1 activates IKK through a helix-loop-helix domain in its N-terminal portion. Simultaneously, Act1 was also cloned by Leonardi et. al., through yeast two- hybrid screening based on its interaction with IKKγ (Leonardi et al., 2000). Likewise,
16 in their study Act1 (referred to as CIKS, connection to IKK and SAPK/JNK) was also found to activate NFκB and JNK.
Initial examination of the amino acid sequence of Act1 revealed a helix-loop- helix domain, which was functionally important for the interaction with IKK, and two TNF-receptor associated factor (TRAF)-binding domains. The TRAF family consists of seven members, TRAF 1-7. Further studies explored the interaction of the
TRAF family members with Act1. CD40, a member of the TNF-receptor superfamily, is expressed on CD4 cells, B cells, and epithelial cells. Upon CD40L stimulation the recruitment of TRAF proteins is necessary for NFκB induction. It was found that upon CD40 stimulation, Act1 is recruited to CD40 and that it also interacts with TRAF3 (Qian et al., 2002). One group reported that over-expression of
Act1 led to the association with TRAF6 through the Act1 TRAF-binding domains, however functional data was lacking (Kanamori et al., 2002). The interaction between
TRAF6 and Act1 was not fully elucidated until recently.
17
Figure 1-2. Act1 structure and IL-17 signaling cascade. (Angkasekwinai et al.)(A) The structure of Act1 consists of two TRAF binding domains that mediate TRAF6 interactions following IL-17 stimulation. Moreover, the U-Box E3 ligase domain is functionally important for mediating the ubiquitination of TRAF6. The helix-loop- helix (HLH) domain and SEFIR domain mediate Act1 protein-protein interactions. Furthermore, the SEFIR domain is necessary for Act1 interaction with IL-17R subunits and for IL-17-dependent NFkB activation. (B) The IL-17A signaling cascade depends on SEFIR-SEFIR domain interaction between the IL-17R subunits and Act1. Following this, Act1 exerts its E3 ligase activity by mediated K63-linked ubiquitination of TRAF6, allowing its interaction with TAK1 and subsequent NFkB activation. IL-17 activation of the ERK, JNK, GSK3 pathways are also Act1 dependent, however the exact mechanism leading to their activation has yet to be elucidated.
STIR domain superfamily
In 2003, Novatchkova et. al. reported on the homology of a protein initially described from zebrafish known as similar expression of fibroblast-growth-factor
18 genes, or SEF (Novatchkova et al., 2003). In the zebrafish SEF acts as an inhibitor of
FGF signaling. Interestingly, they report that in mammals the closest non- orthologous homologue of SEF is found in the IL-17 family of receptors. The sequence of homology in the cytoplasmic region of IL-17R is referred to as the
SEFIR (SEFs and IL-17Rs) domain. Moreover this search also revealed the SEFIR domain was present in Act1.
The SEFIR domain is closely related to the TIR (Toll/interleukin-1 receptors) domain expressed in Toll-like and IL-1 receptors. Due to this similarity, the STIR
(SEFIR and TIR) domain family as it is referred now, consists of the IL-17 receptor subunits including IL-17RA, IL-17RC, IL-17RB, IL-17RD, IL-17RE and Act1. The
STIR domain family provided a foundation implicating the possible involvement of
Act1 in IL-17R signaling. The Act1 and IL-17 interaction was predicted to be analogous to the involvement of the MyD88 adaptor protein in TIR and IL-1 signaling (Figure 2).
ACT1 is a U-box type E3 ubiquitin ligase
Ligand-receptor binding is the initiating factor that sets a series of downstream signaling events into action. Signaling events are dependent on protein interactions and specific modifications. Following the identification of the STIR domain superfamily, Act1 was found to be a critical mediator in the IL-17 signaling pathway.
Studies conducted by Li and Dong revealed the requirement for Act1 in the IL-17 pathway (Chang et al., 2006; Qian et al., 2007). The structure of Act1 provides insight
19 to the mechanism of signal mediation. Taken together the different domains of
Act1—helix-loop-helix domain at the N-terminus, two TRAF-binding domains, and a coiled-coil domain at its C-terminus—suggest protein-protein interactions. It was indeed shown that Act1 is recruited to IL-17R upon IL-17 stimulation through
SEFIR-SEFIR domain interaction, followed by recruitment of the TGFβ Activated
Kinase 1 (TAK1) and TRAF6, leading to NFκB activation. On the other hand, it is intriguing that the IL-17R SEFIR alone was not sufficient to reconstitute IL-17- dependent signaling (Ho and Gaffen, 2010). An additional sequence downstream of the SEFIR was also necessary. It was further shown that the extended SEFIR region in the IL-17RC is required for the interaction with a phosphorylated isoform of Act1, suggesting that the importance of the extended SEFIR region for Act1 modification.
Future studies are required to identify the kinase that is required for Act1 phosphorylation and the relationship with the extended SEFIR region in the IL-17R.
Interestingly, further investigation of Act1 revealed an essential U-box domain that is common in protein E3-ubiquitin ligases. Much like protein phosphorylation, protein ubiquitination is an important modification required for many signaling events. Protein ubiquitination is a sequential process involving the activities of three types of enzymes (E1-E3): the ATP-dependent activity of the ubiquitin-activating enzyme (E1); the acceptance of the activated ubiquitin by the ubiquitin-conjugating enzyme (E2); and an ubiquitin protein ligase (E3), which binds to the E2 and facilitates the conjugation of ubiquitin on the target protein. There are three families of E3 ligases that have been described: RING (really interesting new gene), HECT
20 (homology to E4AP C terminus), and U-box. The conjugation of ubiquitin to different lysine residues can mark proteins for either proteasomal degradation or promote protein-protein interactions if linked on Lys48 or Lys63 respectively.
The study by Lui C. et. al. examined a region within Act1 from residues 273-
338 that is homologous to the U-box domain in other E3-ligases (Liu et al., 2009). In vitro assays revealed that Act1 exhibited E3-ubiquitin ligase activity. Further analysis was aimed at determining the substrate for Act1 ligase activity. It was previously reported that IL-17 signaling and activation of NFκB was dependent on TRAF6
(Schwandner et al., 2000). Upon stimulation with IL-17 ubiquitinated forms of
TRAF6 are detectable in wild-type cells but not in cells lacking Act1. In vitro ubiquitination assays using TRAF6 as a substrate showed that Act1 catalyzed the
Lys63-linked ubiquitination of TRAF6 and that this was dependent on the U-box domain of Act1. Furthermore the ubiquitination of TRAF6 by Act1 was required for the IL-17-dependent activation of NFκB.
The recruitment of Act1 to the IL-17R is through its SEFIR domain and the
U-box E3 ligase activity is the second functional domain of Act1. The U-box domain of Act1 is required for IL-17-dependent activation of NFκB that occurs through the ubiquitination of TRAF6. TRAF6 may not be the only molecule that Act1 can exert its E3 ligase activity and this was suggested in the seminal work by Liu C et al.
Interestingly in this study it was found that in TRAF6 deficient cells, IL-17- dependent activation of NFκB and JNK was abolished however ERK phosphorylation was intact, but this activation was still dependent on the U-box of
21 Act1. These data allude to another downstream component of IL-17-dependent ERK activation that relies on the E3 ligase activity of Act1.
In addition to NF-kB activation, it has also been observed that IL-17 can activate JAK1/2 and PI3K pathway, which coordinates with NFκB activation to upregulate gene expression, especially for host defense genes (e.g. human defensin 2) in human airway epithelial cells (Huang et al., 2007). Moreover, a recent report showed that STAT3 is critical for IL-17-mediated CCL11 expression in human airway smooth muscle cells (Saleh et al., 2009). Furthermore, IL-17 can synergize with
TNFα in the induction of inflammatory gene expression where post-transcriptional effects through mRNA stability play a major role. Interestingly, while Act1 is required for IL-17-mediated stability of KC mRNA induced by TNFα (Hartupee et al., 2007),
TRAF6 is dispensable, implicating other signaling intermediates in mediating IL-17- dependent mRNA stability (Hartupee et al., 2009).
ACT1, the double-edged sword.
Act1 is necessary and sufficient for IL-17-mediated inflammatory responses.
Contrary to its role in the IL-17-dependent responses, Act1-deficiency actually predisposes mice to develop spontaneous autoimmune disease. This observation of spontaneous autoimmune disease in Act1 deficient mice is quite baffling, given that they are resistant IL-17-dependent autoimmune disease models, such as EAE (a mouse model of multiple sclerosis. This intriguing observation adds another layer of complexity to the role of Act1.
22 In fibroblasts, endothelial cells, epithelial cells, astrocytes, and macrophages,
Act1 serves as a component of the IL-17 receptor-signaling cascade. However, in B cells, Act1 serves as a negative regulator of CD40-CD40L and BAFF-BAFFR signaling to control B cell maturation and survival, respectively (Giltiay et al.; Qian et al., 2004; Qian et al., 2002). The loss of Act1 thus results in an increase in B cell population, culminating in splenomegaly, lymphadenopathy, hypergammaglobulinemia, and autoantibody production. In fact, BALB/c mice develop Sjogren-like disease as early as 3wks of age, while C57BL/6 mice exhibit autoimmune phenotype by 9mos of age (Qian et al., 2008). This observation of autoimmune phenotype has also been seen in mice with a spontaneous mutation in the Act1 gene (Matsushima et al.).
In addition to the increase in B cell population, loss of Act1 results in an increased number of Th17 cells. The mechanism of this hyper Th17 response is currently unclear and is still under investigation. However, a possible hypothesis involves Act1’s role in IL-25-mediated signaling and its impact on Th17 cells. First, there is evidence that in the absence of IL-25, there is an increased in Th17 population. Second, treating mice in the EAE model with IL-25 can ameliorate disease severity (Kleinschek et al., 2007). Third, IL-25 can induce the expression of
Th2 cytokines, including IL-13, which has been shown to attenuate Th17 responses
(Newcomb et al., 2009). All of these observations indicate that IL-25 signaling through its receptor has a possible suppressive role on Th17 cells or on the effects of
IL-17 induced inflammatory response. Thus, in the absence of Act1, the loss in IL-25
23 signaling could possibly remove the inhibitory effect on Th17 cells, leading to a hyper inflammatory response. It is exciting to note that three recent independent cohort studies of psoriasis patients found a genetic mutation in Act1 that predisposes them to develop this autoimmune disease (Ellinghaus et al.; Huffmeier et al.; Strange et al.).
Psoriasis is a skin disease characterized by epidermal hyper-proliferation and chronic inflammation of the skin. Th17 cells have been found to be mediators in psoriasis.
Future studies are required to investigate the molecular mechanisms for the precise role of Act1 in modulating Th17 cells and autoimmunity.
One important question is how IL-17 signaling is regulated to adequately control Th17-mediated inflammatory diseases. A recent study showed that TRAF3 is a receptor proximal negative regulator in IL-17 receptor (IL-17R) signaling (Zhu et al.,
2010). TRAF3 greatly suppressed IL-17-mediated signaling pathways and subsequent induction of inflammatory cytokines and chemokines. The binding of TRAF3 to IL-
17R interfered with the formation of the receptor signaling activation complex IL-
17R-Act1-TRAF6, resulting in suppression of downstream signaling. Furthermore,
IL-17 has been found to directly activate ERK-GSK phosphorylation cascades to inactivate C/EBP beta, a critical transcription factor for mediating induction of IL-17 responsive genes (Shen et al., 2009). Future studies are required to elucidate how IL-
17 signaling might be modulated at different levels of its signaling cascades.
These observations in both mouse and human subjects suggest a dual role of
Act1 in modulating the immune response. First, as a component in IL-17 and IL-25- mediated signaling cascades, Act1 serves as a positive role in carrying out
24 inflammatory and allergic reactions. Second, as a negative regulator in B cells, Act1 serves as a keeper that prevents hyper B cell functions to eventually lead to autoimmune phenotypes. Third, due to the complexity of the immune system and the intricate interactions between different cells, receptors, and molecules, Act1’s role as a positive effector in one pathway may lead to its indirect role as a negative effector in another pathway. On one hand, loss of Act1 alleviates autoimmune disease, on the other it exacerbates it. The delicate balance of Act1’s role in the immune system reflects the intricate interactions that are necessary to maintain immune homeostasis and regulate a balance between protection against pathogens and tolerance to self.
25 III. IL-17 and IL-25 associated disease pathology
Pathogenic Th17 cells and Autoimmune Diseases
Both environmental and genetic elements are known to trigger the onset of autoimmunity resulting in the escape of autoreactive lymphocytes from normal selection and the consequent disruption of immune tolerance (Rizzi et al., 2006;
Carson et al., 2006; Cassan and Liblau, 2006; Pagan et al., 2006; Rudensky et al., 2006;
Gauld et al., 2006; Brink, 2006; Cose, 2007). These autoreactive lymphocytes are activated and expanded when they encounter their cognate “self” antigens, infiltrating into the tissues followed by chronic inflammatory responses and tissue destruction, eventually leading to organ-specific (such as multiple sclerosis and type 1 diabetes mellitus) or systemic autoimmune diseases (such as systemic lupus erythematosus and primary sjogren syndrome).
CD4 T helper (Th) lymphocytes play essential regulatory roles in immune responses and autoimmune and inflammatory diseases. Upon activation by professional antigen-presenting cells (APCs), naïve CD4 Th cells differentiate into two subsets: Th1, characterized by production of IFNγ which mediates cellular immunity; and Th2 cells, that synthesize IL-4, IL-5 and IL-13 and functions in humoral immunity and allergic responses. A third lineage of CD4 Th cells, T-helper-
17 (Th17) lineage, has attracted tremendous attention in immunology due to its potent pathogenic role in autoimmune and inflammatory diseases(Cua et al., 2003;
Langrish et al., 2005; Murphy et al., 2003; Harrington et al., 2005; Park et al., 2005;
26 Bettelli et al., 2006; Nakae et al., 2002; Iwakura and Ishigame, 2006; Kolls and
Linden, 2004; Weaver et al., 2006; Harrington et al., 2006; Mangan et al., 2006;
Veldhoen et al., 2006). The pathogenecity of Th17 cells is obviously conferred by the cytokines that they produce, including IL-17A (also referred as IL-17), IL-I7F, IL-21 and IL-22. While IL-17 is the signature cytokine of Th17, recent studies have clearly shown that the other cytokines (IL-21 and IL-22) produced by Th17 cells also play very important roles in the pathogenesis of inflammatory responses(Nurieva et al.,
2007; Caruso et al., 2009; Ma et al., 2008; Zheng et al., 2007).
Th17 and IL-17 in EAE, a murine model for the immunopathogenesis of
MS
Although Th17 cells and IL-17 signaling have been implicated in many inflammatory and autoimmune diseases, experimental autoimmune encephalomyelitis
(EAE) is the best characterized animal disease model for the effector function of
Th17 cells. EAE is an animal model commonly used to study multiple sclerosis
(Stromnes and Goverman, 2006) and can reproduce many of the clinical and neuropathological aspects of the disease. Multiple sclerosis is an autoimmune disease in which self-reactive T-cells specific for myelin antigens elicit an inflammatory response in the central nervous system (CNS) causing damage through demyelination and subsequent axonal injury (Becher et al., 2006; Gold et al., 2006). EAE can be induced by active immunization of animals with myelin antigens or by the adoptive transfer of myelin antigen specific T cells. EAE includes an initiation stage involving
27 activation and expansion of myelin specific T cells in the periphery, an effector stage in which myelin specific T cells in the CNS are reactivated resulting in CNS inflammatory response and a recovery stage in which the immune response is down regulated (McFarland and Martin, 2007; Steinman, 2001).
During the initiation stage, protein antigens (myelin components) and peptides are presented by antigen presenting cells (APCs) within secondary lymphoid organs to the neuroantigen-reactive T cells leading to Th1 and Th17 cell activation and expansion(Cua et al., 2003; Steinman, 2001; Agrawal et al., 2006; Bettelli et al., 2004;
Korn et al., 2009; Yang et al., 2009). Importantly, recent data demonstrate that both
Th1 and Th17 cells can independently induce EAE possibly through different mechanisms. Several studies have suggested that Th1 and Th17 cells might induce distinct types of EAE based on histology and CNS chemokine profile(Lees et al.,
2008; Segal, 2010; Kroenke et al., 2008; Goverman, 2009). Whereas Th2 cells are viewed as counter-inflammatory (Kleinschek et al., 2007), they have been shown to lead to a mild atypical form of EAE (Das et al., 1997; Jager et al., 2009; Steinman,
2008). Nevertheless, Th17 cells are now recognized at least as one of the major mediators in EAE induction and pathogenesis. The disease-relevance of Th17- mediated EAE was underscored by recent data which established a similar relationship between efficacy of IFN-β immunotherapy and Th17 cells in both EAE and MS. Interferon-β (IFN-β), the most commonly-used treatment for MS, is ineffective in a substantial subset of patients for unknown reasons. In EAE, IFN-β reduced EAE signs caused by Th1 cells but exacerbated disease induced by Th17
28 cells. Strikingly, serum from patients who had responded poorly to IFN-b treatment showed high levels of IL-17, while those for whom IFN-b was beneficial demonstrated low serum IL-17 levels (Axtell et al., 2010). These results suggested that
Th17-mediated pathogenesis might define a discrete subset of MS patients for whom
IFN-b does not provide effective disease control.
Recent studies have shown that EAE is substantially reduced in mice lacking
IL-17 or IL-17 receptor and IL-17-specific inhibition suppresses inflammation (Park et al., 2005; Komiyama et al., 2006; Gonzalez-Garcia et al., 2009; Hu et al., 2010).
Similar results were observed in mice deficient in Act1, the key signaling molecule in
IL-17 signaling. Th17 cells are robustly generated in Act1-deficient mice and normally infiltrate the Act1-deficient CNS but fail to recruit hematogenously derived lymphocytes, neutrophils, and macrophages into the CNS. Taken together, these results indicate that whereas IL-17-mediated signaling is not required for the activation of T cells (the initiation stage of EAE), the IL-17-induced Act1-mediated signaling plays an essential role in the effector stage of EAE. While recent studies indicate Th1 cells can also induce EAE, it is important to point out that, in an adoptive transfer experiment, Act1-deficient recipient mice of Th1 cells exhibited similar onset and severity of EAE as wild-type recipients, indicating that Act1 deficiency has no obvious impact on the effector stage of Th1 cell-mediated EAE. In this review, we will further discuss the molecular and cellular mechanism by which
Act1 participates in Th17-mediated effector stage of EAE.
29 To define the effector stage of EAE, a “Two Waves hypothesis” has been proposed. After priming in peripheral lymph nodes, antigen-specific memory
“pioneer” T cells traffic through the choroid plexus into the subarachnoid space
(Wave 1), where they encounter antigen, presented by macrophages (meningeal
APCs), are restimulated and undergo clonal expansion (Ransohoff, 2009; Reboldi et al., 2009). Following expansion, inflammatory cytokines are upregulated and released into the subarachonoid space, acting on adjacent CNS tissue, The ensuing activation of parenchymal vasculature by this cytokine flux allows perivascular leukocyte infiltrates to accumulate (Wave 2) leading to the detrimental inflammatory cascade, a hallmark of the onset of EAE.. One recent study demonstrated how Th17 cells traffic through the choroid plexus into the subarachnoid space (Wave 1). After priming in peripheral lymph nodes, CCR6+ Th17 cells escape from choroid-plexus blood vessels, migrate towards choroid-plexus epithelial cells, which express CCL20 (ligand for CCR6). Signaling through CCR6 allows T cells to cross the tight junctions between the choroid-plexus epithelial cells and enter the cerebral ventricles, from which they migrate to the subarachnoid space. In the first wave, Th17 cells cross the blood-CSF barrier (Ransohoff, 2009; Reboldi et al., 2009) (Fig. 1-2).
30
Figure 1-3. Th17 cells in the Initiation and effector stages of EAE. (A) T cell priming and activation. Protein antigens (myelin components) and peptides are presented by Ag-presenting cells (APCs) within secondary lymphoid organs to the neuroantigen-reactive T cells leading to Th1 and Th17 cell activation and expansion. (B) First wave of Th17 cell entry. After priming in peripheral lymph nodes, CCR6+ Th17 cells escape from choroid-plexus blood vessels, migrate towards choroid-plexus epithelial cells, which express CCL20 (ligand for CCR6). Signaling through CCR6 allows T cells to cross the tight junctions between the choroid-plexus epithelial cells and enter the cerebral ventricles, from which they migrate to the subarachnoid space. Restimulation of T cells by macrophages in the subarachniod space leads to the production of inflammatory cytokines (including IL-17), increased T cell numbers. In summary, in the first wave, Th17 cells cross the blood-CSF barrier. (C) Second wave of Th17 cell entry hypothesized in the proposal. The Th17 cytokines (including IL- 17) from the first Wave impinge on the adjacent CNS tissue to trigger the second Wave of leukocyte infiltration. Astrocytes are perfectly positioned to receive signals from Th17 cells (in the subarachnoid space) and deliver signals associated with conversion of Wave 1 to Wave 2, as their processes comprise the glial limitans adjacent to the meningeal compartment and the same cells extend processes termed astrocyte endfeet, which cover each CNS microvessel. At the onset of Wave 2, after activation of the blood-brain barrier, Th17 cells adhere at the endothelium via upregulated adhesion molecules and begin penetration of the capillary endothelium. Once the activated lymphocytes have extravasated, they are then re-activated by their cognate antigens presented by local APCs in the CNS, leading to the amplification of the inflammatory cascade in the CNS. The large numbers of activated Th17 cells and other inflammatory cells subsequently migrate deeper into the white matter of the
31 CNS parenchyma, resulting in tissue destruction including demyelination and eventually neurologic deficit.
IL-17-induced Act1-mediated signaling in CNS resident cells is necessary for Th17-induced CNS inflammation and EAE pathogenesis
Since Act1 signaling in either endothelial cells or myeloid cells is not the major source contributing to Th17 mediated EAE pathogenesis, the logical next step is to interrogate the IL-17-responsiveness of the CNS resident cells and their participation in EAE development and pathogenesis. Antigen-specific Th17 cells traffic through the choroid plexus into the subarachnoid space where they are reactivated. As a consequence, Th17 signature cytokines, including IL-17, are produced and act on the adjacent CNS tissue. Astrocytes with processes in the glial limitans, and around cerebral blood vessels are positioned to transduce signals from meningeal Th17 cells to activate the BBB endothelium and drive perivascular leukocyte infiltration and concomitant inflammatory cascade associated with the onset of EAE. The large numbers of activated Th17 cells and other inflammatory cells subsequently migrate deeper into the white matter of the CNS parenchyma, resulting in tissue destruction including demyelination and eventually neurologic deficit. Th17 signature cytokines, including IL-17, may directly and/or indirectly act on oligodendrocytes and neurons, leading to demyelination and neurodegeneration. Therefore, it was hypothesized that
IL-17-induced Act1-mediated signaling cascades in different CNS resident cells
(including astrocytes, oligodendrocytes and neurons) coordinately mediate CNS inflammation, demyelination and neurodegeneration, contributing to Th17-mediated
32 pathogenesis of EAE. To test this hypothesis, Act1 was specifically deleted in CNS resident cells by breeding Act1 floxed mice onto Nestin-driven Cre transgenic mice.
The NesCre transgene mediates excision of LoxP-flanked sequences in all neuroectodermal cells of the CNS, including astrocytes, neurons and oligodendrocytes (Graus-Porta et al., 2001; Tronche et al., 1999). Importantly,
NesCre Act1fl/- mice exhibited greatly reduced EAE disease severity induced by active immunization or adoptive transfer of myelin-specific Th17 T cells. Consistent with reduced clinical disease, mononuclear cell infiltrates were substantially decreased in white matter of spinal cords from NesCre Act1fl/- relative to control mice.
Therefore, Act1 deficiency in these CNS resident cells, results in impaired infiltration of inflammatory cells to the CNS, leading to reduced demyelination.
Consistent with the histology, signature IL-17-responsive inflammatory genes
(cytokines, chemokines and matrix metalloproteinases, including CXCL1, CXCL2,
CCL20, CXCL-12, GM-CSF, IL-6, MMP3 and MMP9) was greatly reduced in
NesCre Act1fl/- mice compared to that in control mice. These results suggest that the induction of EAE in CNS-restricted Act1 ablation was impacted by the defective IL-
17 induced inflammatory gene expression. One important question is which CNS resident cell type(s) is responsive to IL-17 and responsible for IL-17-dependent EAE pathogenesis. Astrocytes have been shown to be responsive to IL-17 and play a major role in the production of cytokines and chemokines during EAE. IL-17- and IL-
17/TNF-induced inflammatory gene expression was reduced in Act1-deficient astrocytes as compared to that in control mice. Neurons isolated from wild-type mice
33 showed a very weak response to IL-17 stimulation. These results suggest that the reduction of IL-17 signaling in Act1-deficient astrocytes probably contributes to EAE resistance in CNS-restricted Act1-deficient mice (NesCre Act1fl/-). Astrocytes have direct contacts with the glial limitans as well as the cerebral vasculature, which enables them to couple inflammatory cytokine expression to the infiltration of the CNS by leukocytes. Thererfore, IL-17-induced Act1-mediated inflammatory gene expression in astrocytes might play a critical for the conversion of Wave 1 to Wave 2 during the effector stage of EAE, contributing to Th17-mediated EAE pathogenesis.
IL-17 and IL-25 mediate allergic airway inflammation and airway hyperresponsiveness
Allergic asthma results from a chronic inflammatory response in the lungs with prevailing CD4+ T cells occupying the airways as well as eosinophils and neutrophils, excessive production of mucus and IgE/IgG production. Once activated the CD4+ T-cells differentiate to distinct effector subsets. The CD4+ Th2 cells produce IL-4, IL-5, and IL-13 and act to mediate the humoral and allergic immune responses. In human and in several mouse models of antigen-induced asthma, it is well established that Th2 cells are critical mediators of the immune condition of the lung. However, prior to T-cell activation, resident antigen presenting cells (APC) must present antigens detected at the mucosal surface. Therefore, the mucosal surface is not just a passive barrier but is actually required for the orchestration of an appropriate immune response.
34 Recently, lung epithelium-derived cytokines, IL-25 (IL-17E), IL-33, and TSLP have been found to promote Th2 responses. IL-25 has been demonstrated to promote the differentiation of naïve T-cells to effector Th2 cells in an IL-4 and
STAT6 dependent manner (Angkasekwinai et al., 2007). Additionally IL-25 can also act directly on the epithelial compartment. It was reported that allergens induce the expression of IL-25 in the epithelium and that increased IL-25 expression can promote Th2 immunity (Angkasekwinai et al., 2007). Importantly, intranasal administration of IL-25 leads to an increase in Th2-driving cytokines—IL-4, IL-5, IL-
13, TSLP—and eotaxin and eosinophilia. Although IL-25 is the most divergent IL-17 family member, we and another group found that Act1 interacts with IL-17RB and is required for IL-25-induced responses (Claudio et al., 2009; Swaidani et al., 2009). In the study by Swaidani et. al., Act1 was specifically deleted in the epithelial compartment, which abolished IL-25-induced cytokine production and eosinophilia.
Moreover, it is important to note that in human asthmatic tissue the expression levels of IL-25 as well as IL-25R (IL-17RB) were found to be elevated (Wang et al., 2007).
These studies provide insight into the role of IL-25 signaling in epithelium on the initiation and maintenance of allergic responses (Fig. 1-3).
Importantly, novel tissue-resident innate cell-types have been described as major responders to IL-25 and elicit early Type-2 cytokine response. In fact there are now several innate, non-T not-B–cell-types which include Type-2 innate lymphoid cells (ILC2s), multipotent progenitor type 2 (MPPtype2) cells and Type-2 myeloid
(T2M) cells. Developmentally all of these cell-types arise from the bone-marrow.
35 Upon stimulation with IL-25 (or another epithelial derived cytokine, IL-33) these cells produce IL-4, IL-5 and IL-13. These cells are considered to be an initial source of type-2 cytokines during an allergic response. Indeed ablation of these cell-types results in attenuated allergic asthma responses, or inability to clear helminthic parasite infections.
It has also been shown that IL-17 is also involved in allergic airway inflammation. In contrast to IL-25, when IL-17 is injected to the mouse airway there is a dramatic increase in chemokine expression of KC (CXCL1) and IL-6, which is followed by an accumulation of neutrophils. The administration of IL-17 primarily acts on the epithelial compartment as Act1 deletion from the epithelial compartment leads to an abrogated cytokine and neutrophil response (Fig. 1-3).
Besides mediating the direct induction of distinct airway cellularity and cytokine/chemokine production by IL-17 and IL-25, Act1 is also important in antigen-induced asthma. In the asthma challenge model, mice are immunized with
OVA and are subsequently challenged two weeks later with OVA aerosol. Mice that are deficient in Act1 in the epithelial compartment have reduced airway eosinophilia/neutrophilia and reduced cytokine/chemokine production. It is important to note that there is no difference in OVA-specific IgE or IgG production or airway hyperresponsiveness in the epithelial-deleted Act1 mouse. This observation may be due to the essential role of Act1 in other immune cell types, namely T-cells.
Overall, these findings demonstrate the utility of a common signaling component,
Act1, and its role in the epithelial compartment. How Act1 mediates the diverse
36 immune response by IL-17 and IL-25 is yet to be determined, but most likely it may be explained by the specific signaling events mediated by different receptor subunits.
Figure 1-3. IL-17 and IL-25 in airway inflammation. (Angkasekwinai et al.)(A) IL- 17 is produced by CD4+ Th17 cells. IL-17 stimulates the airway epithelium to produced chemokines such as Cxcl1, CSF2 and CSF3 which promote the recruitment of neutrophils and macrophages to the airways. (B) In response to inhaled allergens, bacteria or fungi, stimulation of protease activated receptors (PARs) or toll-like receptors (TLRs) leads to the expression of IL-25 from the airway epithelium. IL-25 can further stimulate naïve- or Th2 CD4+ T-cells, or innate lymphoid cells (ILC) to produce type-2 cytokines, Il-4, Il-5 and Il-13. Furthermore IL-25 may also stimulate cytokine production from the epithelium, including its own expression, thereby creating an amplification loop. Ultimately IL-25 leads to eosinophil recruitment and antibody production (IgE).
37
IV. IL-17 and IL-25 signaling
TNF receptor-associated factor (TRAF) protein family
TNF-receptor associated factors (TRAFs) are a family of proteins that were initially identified based on their interactions with members of the Tumor Necrosis
Family (TNF) of Receptors (TNFRs). There are 7 members of the TRAF family, termed TRAF1 through TRAF7, which, with the exception of TRAF7, are each characterized by a highly conserved C-terminal TRAF domain. Importantly, TRAFs
2-5 all have a RING (Really interesting new gene) finger domain at the N-terminus of the protein. The RING domain facilitates binding to ubiquitin conjugating molecules known as E1 and E2 proteins. Further the RING-domain containing TRAF molecules bind to target proteins and thus facilitate ubiquitin conjugation on the recognized target thereby functioning as an E3-ubiquitin ligase. Although each TRAF protein has the RING-domain, only TRAF2 and TRAF6 have been demonstrated to exhibit E3-ligase activity.
The TRAF1, 2, 3, 5, 6 family members have each been implicated in numerous signaling pathways. TRAF1 and TRAF2 are recruited to the TNF-R upon ligand stimulation and are required for ubiquitination and subsequent activation of
NFkB. CD40 is a TNF-R family member expressed on B-lymphocytes and upon
CD40L binding, the resting B-cell becomes activated, proliferates and begins generating antibodies. TRAF proteins 1, 2, 3, 5, 6 have all been implicated in CD40 signaling. Although TRAF6 is not required for TNF-R signaling, it is a crucial TRAF
38 molecule linking receptor-mediated NFkB activation. Notably interleukin-1-Receptor
(IL-1R) and toll-like receptor (TLR) engages TRAF6 through the activation of interleukin-1 associated kinases (IRAKs). TRAF6 then mediates K63-linked ubiquitination of IκBα kinase (IKK), which is required for IκBα degradation and
NFkB nuclear translocation . Moreover, TRAF6 is also required for NFkB activation downstream of the IL-17R family (Schwandner et al., 2000).
Unlike the other TRAF proteins, TRAF4 does not actively participate in TNF-
R, CD40, IL-1R or TLR signaling pathways. Several structural differences set it apart from the other TRAF family members. First, while TRAF1, 2, 3, 5 contain the three key residues R, Y and S, which are required for recognition of the TRAF interacting motif (TIM) of the TNF-R, these are not conserved in TRAF4 (Hornung M et al.,
1998; Kedinger and Rio 2007). Second, TRAF4 is the only TRAF family member with a putative nuclear localization sequence (NLS). The function of the NLS has not been carefully examined. Lastly, TRAF4 contains seven zinc finger domains, whereas the other TRAF proteins only have six.
There is little known about the involvement of TRAF4 in cellular signaling pathways. Currently TRAF4 has been implicated as a negative regulator of TRAF6- dependent NFkB activation in inflammatory pathways, including TLR4- and NOD2
(Takeshita et al., 2005;Marinis et al., 2010). Studies in drosophila larvae, xenopus larvae and mice suggest that TRAF4 has a role in developmental pathways. For instance, in drosphila embryo, TRAF4 is upregulated during apical constriction of the mesoderm by the transcription factor, Twist and TRAF4 was shown to associate with
39 Armadillo (β-catenin) and modulate its apical positioning (Mathew et al., 2011). In mice, TRAF4-deficiency leads to developmental abnormalities depending on the genetic strain. On a mixed 129/Svj X C57BL/6 background TRAF4-deficiency results in a constricted tracheal ring at the larynx junction (Shiels et al, 2000).
However on 129/Svj background, TRAF4-deficiency is lethal for approximately 1/3 of the mutant pups. Surviving mice exhibit normal immune systems, however they do exhibit altered locomotion coordination typical of ataxia (Regnier et al., 2002).
Although the genetic differences accounting for this lethality have yet to be defined, it is clear that TRAF4 can participate in early developmental pathways.
Figure 1-5. TNF-receptor Associated Factor (TRAF) Family. All TRAF family members are characterized by a TRAF-domain in the C-terminus. The TRAF-domain facilitates TRAF-TRAF interaction and dimer- or oligo-merization during cell
40 signaling processes In the mid-region is a coiled-coil domain and zinc-finger repeats, both of which mediate protein-protein interactions. At the N-terminus is RING finger domain which confers E3-ligase activity to TRAF’s denoted with an *.
IL-17 and IL-25 signaling pathways, a current overview
Traditionally in TNFR and toll-like receptor pathways, TRAF proteins are activated after ligand stimulation. Following this it is thought that TRAFs, such as
TRAF6, recognize downstream targets such as IKK that is ubiquitinated leading to the activation of NFkB. For the IL-17 pathway, ACT1 is the immediate E3-ligase activated following ligand stimulation. TRAF-binding domains within the ACT1 secondary structure leads to specific TRAF engagement. For instance, as mentioned earlier, ACT1 via its E3-ligase activity can bind to and ubiquitinate TRAF6 (Liu C et al., 2009). This event leads to TRAF6-dependent NFkB activation via TAK1-IKKα/β complex activation. TRAF6 was also identified as critical mediator of NFkB activation in the IL-25 signaling pathway (Maezawa et al., 2006). Although the exact mechanisms were not explored, due to IL-25’s dependency on ACT1, the engagement of TRAF6 is probably via ACT1-dependent mechanism.
Upon IL-17 binding to its cognate receptor, ACT1 is recruited to the receptor complex where it can be phosphorylated. The phosphorylation of ACT1 leads to a dramatic shift in ACT1 mobility in SDS-PAGE gels. Mass spectrometry analysis of the modified ACT1 revealed a distinct phosphorylation at Serine position 311 (Bulek et al., 2011). Interestingly, specific mutation of this serine did not alter NFkB activation but rather diminished IL-17’s capacity to stabilize mRNA. Indeed IL-17 induced stabilization of mRNA is dependent upon ACT1 binding to another
41 complex of TRAF molecules, composed of TRAF2 and TRAF5 (Sun et al., 2011). In silico modeling revealed that phosphorylation of the Serine-311 by the inducible kinase IKKi (or IKK epsilon) actually altered ACT1’s TRAF affinity, shifting from
TRAF6 towards an affinity for TRAF2/TRAF5. Importantly, downstream RNA- binding proteins are sequestered by TRAF2/TRAF5, such as splicing factor-2 (SF2) a de-stabilizing RNA binding protein, in exchange for HuR (human antigen R) a stabilizing one, to extend the half-life of chemokine mRNA (Herjan et al., 2013).
In addition to the TRAF-binding domains on ACT1, the IL-17RA subunit also contains a putative TRAF-binding site. This site leads to preferential occupation by another TRAF molecule, TRAF3. Binding of TRAF3 actually diminishes IL-17 responses (Zhu et al., 2010). For instance, in TRAF3-null cells, IL-17 signaling and gene expression are significantly enhanced. Furthermore, in IL-17-driven disease models, disease pathogenesis is actually worse when TRAF3 is ablated. Therefore, in addition to positive actions, TRAF proteins also exert a negative feedback control to tune the IL-17A response.
Together these data imply that distinct TRAF molecule engagements mediate specific downstream signaling outcomes. The exact mechanisms that determine which TRAF is activated remains to be fully elucidated. However, based on the findings from Bulek et al., ACT1 phosphorylation by IKKi, was a critical initiating event that provoked the TRAF2/TRAF5 associations and subsequent stabilization of chemokine mRNA. In addition to phosphorylation, ACT1 has been reported to be ubiquitinated during prolonged IL-17 treatment in cultured cells. This ubiquitination
42 event promoted ACT1 degradation, by K48 linkage by the E3 ligase β−TRCP (Shi et al., 2011). This observation however may suggest that other non-degrading K63- linked ubiquitination events may also be possible. Moreover, as another mechanism linking specificity to TRAF engagement, there is much evidence that TRAF molecules are expressed in a tissue-specific manner. Thus relative abundance of certain TRAF molecules in the IL-17-responsive cell-type may delineate the types of responses.
Figure 1-6. IL-17 and IL-25 signaling. IL-17A binds to IL-17RA and IL-17RC, which mediate the recruitment of ACT1 through homotypic SEFIR-SEFIR interactions. ACT1 then engages TRAF proteins for subsequent target gene transcription. IL-25 binds to IL-17RA and IL-17RB, which facilitate ACT1 recruitment via the SEFIR domains. TRAF6 is required for IL-25-induced NFkB activation. IL-25 stimulation in epithelial as well as in T-cells has been shown to
43 activate MAPK, PI3K-AKT and upregulates the expression of transcription factors GATA3, NFAT2 and JunB (Angkasekwinai et al., 2007). Through unknown mechanisms IL-25 can stimulate the transcription of a distinct subset of genes associated with type-2 immunity.
44
Chapter 2:
TNF Receptor-Associated Factor 4 Restricts IL-17- mediated pathology and signaling processes
Jarod A. Zepp1,2, Caini Liu1, Wen Qian1 , Ling Wu1,3, Muhammet F. Gulen1, Zizhen Kang1 and Xiaoxia Li1,2,3
1Department of Immunology, Cleveland Clinic, Cleveland, OH, USA: 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. 3Department of Pathology, Case Western Reserve University
Portions of this study were published in Zepp JA, et al. Cutting Edge: TNF Receptor- Associated Factor 4 restricts IL-17-mediated pathology and signaling processes. J. Immunol. 2012, 189 (1):33-7. With permissions from The Journal of Immunology (Appendix 3)
Copyright © 2012. The American Association of Immunologists, Inc.
45 I. Abstract
The effector T-cell subset, Th17, plays a significant role in the pathogenesis of
multiple sclerosis as well as other autoimmune diseases. The signature cytokine, IL-
17, engages the IL-17R and recruits the E3-ligase Act1 upon stimulation. In this study
we examined the role of TRAF4 in IL-17 signaling and Th17-mediated autoimmune
encephalomyelitis. Primary cells from TRAF4-deficient mice displayed markedly
enhanced IL-17-activated signaling pathways and induction of chemokine mRNA.
Adoptive transfer of MOG 35-55 specific wild-type Th17 cells into TRAF4-deficient
recipient mice induced an earlier onset and a prolonged course of disease.
Mechanistically, we found that TRAF4 and TRAF6 utilized the same TRAF-binding
sites on Act1, allowing the competition of TRAF4 with TRAF6 for the interaction
with Act1. Taken together, this study reveals the necessity of a unique role of TRAF4
in restricting the effects of IL-17 signaling and Th17-mediated disease.
46 II. Introduction
IL-17 (IL-17A) is the signature cytokine of Th17 cells, a recently defined
effector T cell lineage that exhibits a distinct differentiation program from classical
Th1 and Th2 cells. IL-17 has been linked to the pathogenesis of numerous
autoimmune and inflammatory diseases. IL-17 levels are elevated in many
inflammatory conditions, such as multiple sclerosis, rheumatoid arthritis,
inflammatory bowel disease, asthma and psoriasis. IL-17-deficient mice are resistant
to experimental autoimmune encephalomyelitis (EAE) (Komiyama et al., 2006) and
collagen-induced arthritis (Nakae et al., 2003) compared to that of wild-type mice,
indicating the essential role of IL-17 signaling in these inflammatory conditions. The
major function of IL-17 is to coordinate local tissue inflammation by promoting
production of inflammatory cytokines, chemokines and matrix metalloproteinases,
resulting in the infiltration of inflammatory cells such as neutrophils, monocytes and
lymphocytes.
The IL-17 receptor family consists of five members, of which, IL-17A and IL-
17F signal through a heterodimeric receptor complex composed of IL-17RA and IL-
17RC (Hu et al., 2010). Both IL-17RA and IL-17RC belong to a SEF/IL-17R
(SEFIR) protein family, which is defined by the presence of a conserved cytoplasmic
SEFIR domain (Novatchkova et al., 2003). Act1/CIKS (NF-κB-activator-1) is an
essential component in IL-17 signaling and is required for IL-17-dependent immune
responses (Chang et al., 2006;Qian et al., 2007). Act1 is also a member of the SEFIR
protein family, containing a SEFIR domain at its C-terminus. Upon IL-17
47 stimulation, Act1 is recruited to IL-17R through a SEFIR-dependent interaction.
Furthermore, Act1 possesses a U-Box domain that is functionally required for its E3- ligase activity (Liu et al., 2009). Upon IL-17 stimulation Act1, together with the
Ubc13-Uev1A E2 complex, exerts K63-linked polyubiquitination of TRAF6. This ubiquitination event is required for TRAF6-mediated activation of TAK1 and the
IKK complex, resulting in activation of NF-kB and subsequent NF-kB-dependent transcription of pro-inflammatory and neutrophil-mobilizing cytokines and chemokines. Furthermore, IL-17 also activates MAPK pathways such as ERK, P38 and JNK. Additionally, IL-17 can promote the stability of numerous mRNA encoding cytokines and chemokines in a TRAF6-independent manner (Hartupee et al., 2007;Hartupee et al., 2009). Recently, we reported that following IL-17 stimulation, a phosphorylated form of Act1 forms a complex with TRAF2 and
TRAF5 (Bulek et al., 2011;Sun et al., 2011). This phosphorylation event and complex formation are functionally required for the stabilization of KC and G-CSF mRNA.
While IL-17 is required for host defense against extracellular microorganisms, it is also involved in the pathogenesis of several autoimmune diseases. Therefore, it is critical to explore any mechanisms that possibly regulate and restrict this pathway.
It has been shown that IL-17 induces ERK- and GSK3b-dependent phosphorylation of C/EBPb restricting the expression of proinflammatory genes (Ruddy et al.,
2004;Shen et al., 2009). We recently showed that TRAF3 is a crucial negative regulator of IL-17R–mediated signaling (Zhu et al., 2010). Binding of TRAF3 to IL-
17R interferes with the formation of the positive signaling complex IL-17R–Act1–
48 TRAF6, hence dampening IL-17-induced signaling and inflammation. In the present
study, we identified another TRAF member, TRAF4 as a novel negative regulator in
IL-17-mediated signaling and inflammatory responses via a distinctive mechanism.
TRAF4 interacted with Act1 in an lL-17-dependent manner, and competed with
TRAF6 for the same TRAF-binding sites on Act1, thereby reducing formation of
Act1-TRAF6 signaling complex. Consistently, while TRAF4 deficiency increased the
IL-17-induced inflammatory responses in vitro and in vivo, overexpression of
TRAF4 suppressed IL-17-mediated signaling and gene expression. Our results reveal
TRAF4 as the first Act1-interacting partner negatively regulating IL-17 signaling and
implicate TRAF4 as a potential novel target for clinical intervention of IL-17–
mediated autoimmune disorders.
III. Materials and Methods
Mice, cell culture and reagents
TRAF4-deficient C57/BL6 mice were generated as described previously (Shiels et al.,
2000). Mice were housed in a temperature-controlled facility on a 12-h light cycle.
Experiments were performed with gender-matched mice aged 6-8 weeks. The
Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic
Foundation approved all animal procedures. Hela, MEF and HEK293 cells were
maintained in DMEM, supplemented with 10% FBS, penicillin G (100 µg/ml) and
streptomycin (100 µg/ml). Primary mouse astrocytes were isolated as described
previously (Qian et al., 2007) and maintained in DMEM supplemented with 10%
FBS. Isolation of primary kidney cells was described previously (Bulek et al., 2011).
49 Briefly, kidneys from 3-week old mice were removed, minced and digested with 0.5%
Trypsin at 37C for 30 minutes and this was repeated three times. Between each incubation period, the supernatant was collected into tubes and placed on ice. Then the trypsin digestion was spun down and resuspended in DMEM with 10% FBS.
Omni-tagged TRAF4 expression vector was provided by Derek Abbott, Case
Western Reserve University, described in ref. (18). Antibodies to phosphorylated-
ERK1/2, JNK, IkBa and total IkBa were from Cell Signaling Technology. Goat anti-
TRAF4 and the TRAF6, TRAF3, and hAct-1 (all rabbit) antibodies were from Santa
Cruz Biotechnology and the rabbit anti-TRAF4 antibody was from Epitomics. The antibodies to TAK1 and Act1 were described previously (Qian et al., 2007;Xiao et al.,
2008). The murine-rIL-17A and human-rIL-17A were from R&D Systems.
Coimmunoprecipitation and transfection
Act1-deficient MEF cells were reconstituted with stable expression of wild-type Act1, described in (Liu et al., 2009). The cells were grown to 80% confluence in 15 cm flat- bottom tissue-culture dishes. Fresh medium was given to the cells the day before IL-
17 stimulation. Following stimulation the media was removed and the cells were rinsed with ice-cold PBS and then gently scraped from the plate into ice-cold PBS.
The cells were pelleted in 4-degree centrifuge (2000 xRPM for 5 minutes) and the pellet was lysed in Co-IP buffer (0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM b-glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Roche). Antibodies were conjugated
50 to Protein-A or Protein-G sepharose beads. The cell lysate was clarified by high- speed centrifugation and then applied to the antibody-conjugated beads and then placed on a rotator overnight. The following day the beads were washed 5-times with ice-cold Co-IP buffer, then Laemmeli buffer (BioRad) was added, then the samples were separated by SDS-PAGE and analyzed by immunoblotting. For transient transfection of HELA cells, 5 µg of each Omni-tagged human TRAF4 were mixed along with Lipofectamine-2000 (Invitrogen) according to the manufacture’s instructions.
Induction and assessment of EAE
Passive EAE was induced and assessed as previously described (Qian et al.,
2007;Kang et al., 2010). Briefly, TRAF4-deficient of littermate control mice were injected with polarized MOG 35-55-specific Th17 cells 4 hours after 500 Rad sublethal irradiation. MOG-specific Th17 cells were generated by collected draining lymph node and spleen cells from wild-type (Taconic Farms) 10 days after immunization. Cells were culture for 5 days with MOG 35-55 (25 µ g/ml) with 20 ng/ml rmIL-23 for Th17 cell polarization.
Isolation and analysis of CNS inflammatory cells
Brains were collected and homogenized in ice-cold tissue grinder in ice-cold RPMI.
The cells were collected by centrifugation at 1200 x RPM for 5 minutes at 4C. Cells were then resuspended in 10 ml of 30% Percoll (Amersham Bioscience) and applied
51 over a 70% Percoll cushion in 15 ml tubes at 800 x g for 30 minutes. Cells were collected from the 30-70% interface and subjected to flow cytometry. Fluorescence- conjugated CD4, CD8, CD11b, CD45, Ly6G antibodies and isotype controls were purchased from BD Biosciences and the F4/80 antibody was from Serotech.
Real-time PCR
Total RNA was extracted from primary kidney cells with TRIzol reagent (Invitrogen) and isolated according to the manufacturer’s instructions. Total RNA from spinal cord sections was collected by column purification using the MiRVana RNA isolation kit (Applied Biosystems). All gene expression results are expressed as fold-change normalized to the expression of β-actin and the untreated control. Fold-induction of gene expression in the spinal cords is relative to naïve/unchallenged TRAF4-deficient and littermate control spinal cord RNA. Primer sequences have been previously described (Kang, 2010 #14).
Statistics
The p values of clinical scores were determined by one-way multiple-range analysis of variance (ANOVA) for multiple comparisons. Other p-values were determined by
Student’s t tests (two-tailed).
52 IV. Results
IL-17-induced signaling and gene expression are increased in TRAF4-deficient
cells
Following IL-17 stimulation and Act1 recruitment, TRAF family members
TRAF-2, -5 and -6 are required for NF-κB activation and mRNA stability. On the
other hand, TRAF3 interacts with the IL-17R and suppresses Act1 and TRAF6
recruitment. One important question is whether TRAF4 plays any role in IL-17-
mediated signaling. To test this, we isolated primary kidney epithelial cells, which are
highly responsive to IL-17 treatment (Bulek et al., 2011;Sun et al., 2011), from
TRAF4-deficient and littermate control mice. Both basal level and IL-17-induced
phosphorylation of ERK1/2, JNK and IkBa were markedly increased in the TRAF4-
deficient cells compared to that in the control cells (Fig. 2-1 A). Furthermore, IL-17-
induced IkBa degradation was significantly enhanced in the absence of TRAF4 (Fig.
2-1 A). To recapitulate these data, we employed another system in which we
transduced human cervical carcinoma cell line (Hela) cells with retrovirus encoding
human-TRAF4. Importantly, in these TRAF4-overexpressing cells, the IL-17-induced
phosphorylation of ERK1/2, JNK and IkBa and degradation of IkBa were reduced
(Fig. 2-1 B). Furthermore, siRNA directed at Traf4 or overexpression of TRAF4 had
a similar effect in Hela and MEF cells (Fig. 2-1 C,D). Taken together, these results
indicate that TRAF4 expression can suppress IL-17-induced signaling events.
We next examined the impact of TRAF4 deficiency on IL-17-induced gene
expression. The IL-17-induced expression of CXCL1 (KC), G-CSF and IL-6 was
53 substantially increased in TRAF4-deficient primary kidney epithelial cells as compared to that in control cells (Fig. 2-1 E). Importantly, although the basal levels of G-CSF were lower in the TRAF4-deficient cells, the fold induction of G-CSF in response to
IL-17 stimulation was greater than that in control cells. These observations suggest a suppressive role of TRAF4 in IL-17-mediated inflammatory gene expression.
54
Figure 2-1. IL-17 induced signaling and gene expression in TRAF4-deficient primary cells. Primary kidney cells were isolated from 3-week old TRAF4-dificient mice and littermate controls. (A) Cells were stimulated for 0, 15, 30 and 60 minutes with mIL- 17A (50 ng/ml). Cell lysate was made and Immunoblot for the indicated proteins was performed. (B) Hela cells were transduced with retrovirus encoding hTRAF4. Cells were stimulated for the indicated times with hIL-17A, cell lysate was collected and subjected to Immunoblot. For immunoblots, one representative blot is shown from 4 (A) or 3 (B) independently performed experiments.
55
C D
E
Figure 2-1. IL-17 induced signaling and gene expression in TRAF4-deficient primary cells. (C) Western analyses from Hela cells transfected with siRNA directed against human- TRAF4 and stimulated with IL-17 (50 ng/ml) for the indicated times. (D) Murine MEF cells were transfected with TRAF4 or control vector and stimulated with IL-17 for the indicated times. Results shown are from one representative experiment repeated two times. (E) Primary kidney cells from TRAF4-deficient mice and littermate controls were stimulated for the indicated times with IL-17. Total RNA was collected and cDNA was made after which quantitative real-time PCR was performed with specific primers for KC, G-CSF and IL-6, n=5, means ± SEM, *p<0.05, **p<0.01 for control versus stimulated. For immunoblots, one representative blot is shown from 4 (A) or 3 (B) independently performed experiments.
56 TRAF4 deficiency exacerbates EAE severity
Previous studies have shown that IL-17 signaling is essential in the development of experimental autoimmune encephalomyelitis (EAE). We have recently reported that Act1 deficiency rescues mice from disease pathology in EAE
(Kang et al., 2010). Although TRAF4 deficiency enhanced IL-17 signaling in cell culture, we utilized a Th17-cell mediated EAE model to determine the in vivo impact of TRAF4 deficiency. We generated MOG-specific Th17 cells from wild-type
C57/BL6 mice and then transferred them into irradiated TRAF4-deficient and littermate control mice. The TRAF4-deficient mice displayed an earlier onset of disease compared to littermate controls. Furthermore the peak severity of disease was prolonged in the TRAF4-deficient mice (Fig. 2-2 A-B). Next, we examined the cell infiltrates in the brains of TRAF4-deficient and littermate control mice at the peak of disease. Consistent with the clinical severity of disease, the TRAF4-deficient mice had increased numbers of immune cell infiltration in the brain (Fig. 2-2 C). Furthermore, the TRAF4-deficient mice had significantly higher expression of pro-inflammatory genes as compared to that in the control mice (Fig. 2-2 D).
We previously reported that Act1-deficient mice are resistant to EAE.
Moreover the cell-specific contribution of Act1-deficiency in the development of
Th17-mediated EAE, revealed that Act1 expression in the CNS resident cells, specifically in astrocytes, was necessary for induction of EAE (Kang et al., 2010).
Thus we examined IL-17-dependent gene induction in TRAF4-deficient astrocytes.
Following IL-17 stimulation the TRAF4-deficient astrocytes exhibit markedly
57 increased expression of CXCL1, GM-CSF and IL-6 compared to the heterozygous control (Fig. 2-2 E). Taken together, these data suggest that while TRAF4 expression can attenuate IL-17-mediated signaling events, lack of this suppressive effect of TRAF4 in vivo results in an accelerated and more severe EAE.
Figure 2-2. TRAF4 deficiency exacerbates Th17 mediated EAE. (A) Serum cytokine concentration in TRAF4 deficient mice following intraperitoneal administration of IL-17 (2 µg/mouse for two consecutive days).
58
Figure 2-2. TRAF4 deficiency exacerbates Th17 mediated EAE. Wild-type C57/BL6 mice were immunized with MOG-peptide (35-55) for 10 days. After which spleen and lymph node cells were harvested and re-stimulated with rmIL-23 (20 ng/ml) and MOG-peptide. The Th17 polarized cells were transferred into irradiated TRAF4- deficient mice and littermate controls. (B) Following adoptive transfer of MOG- specific Th17 cells, clinical severity of EAE was scored (materials and methods).
59
Figure 2-2. TRAF4 deficiency exacerbates Th17 mediated EAE. (C) At the peak of disease, brains were homogenized and infiltrating immune cells were analyzed by flow-cytometry. Total cell numbers were calculated based on frequency of the indicated marker in the total cell population. (D) Spinal cords were harvested at peak of disease and total RNA was isolated and cDNA was made. Calculated fold-change of the indicated cytokine/chemokines was relative to naïve TRAF4-deficient and littermate control spinal cord RNA. (E) Gene expression from primary astrocytes stimulated with IL-17 (50 ng/ml) for 16 hours. For (D-E) quantitative real-time PCR was performed with specific primers for the indicated cytokines/chemokines relative to b-actin. n=5 per group for (D) and n=2 (E), means ± SEM, *p<0.05, **p<0.01 for littermate heterozygous control versus TRAF4-deficient. For adoptive transfer experiments n=5 per group, shown here is one of two independently performed experiments, ***p<0.001 determined by one-way ANOVA.
60 Act1/TRAF4 complex is distinct from other Act1/TRAF complexes
Considering the importance of TRAF4 in modulating IL-17 signaling, we examined TRAF4 complex formation in response to IL-17 stimulation. We first examined whether Act1 interacts with TRAF4 in response to IL-17 stimulation. We immunoprecipitated Act1 from cell lysates of untreated and IL-17-treated MEFs, followed by western analysis with antibodies against different TRAFs and TAK1. As shown in Figure 2-3 A, IL-17 stimulation indeed induced the interaction of Act1 with TRAF4, in addition to its interaction with TRAF6, TRAF3 and TAK1. Next, we assessed whether TRAF4 is associated with TRAF6 and TAK1 as the Act1-TRAF6-
TAK1 complex is essential for IL-17-induced NF-kB activation. Interestingly, through co-immunoprecipitation with anti-TRAF6, we found that neither TRAF3 nor TRAF4 was associated with the IL-17-induced TRAF6 complex (Fig. 2-3 B).
Consistently, Act1 but not TRAF3, TRAF6 or TAK1 was detected in the TRAF4 immunoprecipitate from cell lysates of IL-17-treated MEFs (Fig. 2-3 C). Taken together, these data indicate that the IL-17-induced Act1-TRAF4 complex is distinct from other Act1/TRAF complexes (Act1-TRAF6 and Act1-TRAF3).
61
Figure 2-3. Distinct Act1-TRAF interactions form following IL-17 stimulation. Act1- deficient MEFs restored to express constitutive wild-type Act1 were stimulated with IL-17A for the indicated times. (A) Following stimulation with IL-17, cells were lysed in Co-IP buffer and immunoprecipitation against Act1 (A), TRAF6 (B) and TRAF4 (C) was performed. The immunoprecipitates and whole cell lysate (WCL) were subject to SDS-gel electrophoresis and Immunoblot analysis was performed for the indicated proteins. Shown here are single experiments that are representative of three independently performed assays.
62
TRAF4 competes with TRAF6 for Act1 TRAF-binding-sites
One important question is how the Act1/TRAF4 complex attenuates IL-17- mediated signaling events. Since enhanced IkBa phosphorylation and degradation were observed in TRAF4-deficient cells, we focused on the mechanistic role of
TRAF4 in modulating NF-κB pathway. We previously reported that Act1 binds to
TRAF6 and exerts E3-ligase activity in response to IL-17 stimulation, resulting in
K63-linked polyubiquitination of TRAF6 (Liu et al., 2009). This modification event is necessary for the interaction of TRAF6 with TAK1 and subsequent NF-κB activation
(Liu et al., 2009). Since Act1-TRAF4 and Act1-TRAF6 are two distinct complexes, it is possible that TRAF4 competes with TRAF6 for the binding with Act1 so that
TRAF4 deficiency enhances the IL-17-induced association of Act1 with TRAF6 and consequent NF-kB activation. To test this we immunoprecipitated TRAF6 from cell lysates of untreated and IL-17-treated TRAF4-deficient and control primary kidney cells.. Consistently, there were markedly enhanced modified TRAF6 bands in the
Act1 immunoprecipitates from IL-17-treated TRAF4-deficient cells compared to that in the control cells (Fig. 2-4 A). Indeed we observed increased association of Act1 with TRAF6 in the TRAF4-deficient cells compared to that in control cells (Fig. 2-4
B). These data suggest that TRAF4 deficiency resulted in more Act1-TRAF6 interaction and TRAF6 modification (previously shown as ubiquitination). To further corroborate these studies we stably expressed TRAF4 in MEFs and immunoprecipitated TRAF6 to assess Act1 binding. TRAF6/Act1 interaction was
63 reduced when TRAF4 was overexpressed (Fig. 2-4 C). Taken together these results support our hypothesis that TRAF4 may indeed compete with TRAF6 for binding to
Act1, thereby inhibiting Act1-mediated TRAF6 ubiquitination and subsequent NF-kB activation.
We previously reported that Act1 contains two putative TRAF-binding sites, both of which are required for binding to TRAF6 (Liu et al., 2009). We then tested whether TRAF4 competes with TRAF6 for the binding to Act1 through these
TRAF-binding sites. Indeed increasing amounts of TRAF4 could compete out the
TRAF6/Act1 interaction (Fig. 2-4 D). Next, Act1-TRAF4 interaction was examined in Act1-deficient MEFs that have stable expression of Act1-wild-type and Act1-TBm
(TRAF-domain-site-1-site-2 mutant Act1). Through immunoprecipitation of TRAF4 we observed that interaction was limited to Act1-wild-type but it was abolished in the
Act1-TBm (Fig. 2-4 E), demonstrating that TRAF4 and TRAF6 interact with Act1 via the same TRAF-binding domains. Taken together, these results provide the molecular basis for the observed competition between TRAF4 and TRAF6 for their interaction with Act1 (Fig. 2-5).
64
Figure 2-4. TRAF4 restricts Act1/TRAF6 interaction. Primary kidney cells were isolated from TRAF4-deficient mice and littermate controls. Cells were treated with IL-17 (50 ng/ml) for the indicated times and immunoprecipitation of Act1 (A) and TRAF6 (B) was performed as well as Rabbit-IgG control was included.
65
D
Figure 2-4. TRAF4 restricts Act1/TRAF6 interaction. (C) MEFs were transfected with TRAF4 or vector control as indicated. After 48 hours cells were stimulated with IL- 17 (50 ng/ml) for the indicated times, cell lysate was collected in Co-IP buffer and immunoprecipitation against hAct1 was performed. Cell lysate were subjected to Immunoblot analysis for the indicated proteins. (D) HEK293 cells were transfected with the indicated plasmids followed by co-immunoprecipitation against myc-tag Act1.
66 E
Figure 2-4. TRAF4 restricts Act1/TRAF6 interaction. (E) Act1-deficient MEF’s were reconstituted with wild-type Act1 or Act1-TBm. These cells were stimulated with IL- 17 (50 ng/ml) for the indicated times and cell lysate was collected in Co-IP buffer. TRAF4 (top) or Act1 (bottom) was immunoprecipitated from the cell lysate and then subjected to Immunoblot analysis for the indicated proteins. Shown here are immunoblots from single experiments that are representative of three independently performed assays.
67
Figure 2-5. Working model of IL-17 signaling cascade. (0) Represents the resting or an unstimulated cell (1) IL-17 stimulation engages IL-17RA and IL-17RC (2) Act1 is recruited to and interacts with the receptor via the SEFIR domain (2-3) These events represent TRAF-binding to Act1, TRAF4 restricts Act1/TRAF6 interaction (4) Modified Act1 will promote mRNA stability (5) Downstream activation of NF-κB, mRNA stability or additional events will occur.
68
Discussion
Since the discovery that Act1 is the essential component for IL-17 signaling, several TRAF molecules have been shown as direct interaction partners of Act1 and play important roles in IL-17-mediated signaling pathways. TRAF6 is a direct substrate of Act1 U box E3 ligase and its ubiquitination is critical for IL-17-induced
NF-κB activation. TRAF2 and TRAF5 are also recruited to Act1 to mediate IL-17- induced mRNA stabilization. In the present study, we identified another member of the TRAF family, TRAF4, as a novel interaction partner of Act1 thus forming a unique signaling complex upon IL-17 stimulation. IL-17-induced signaling and gene expression were greatly enhanced in the TRAF4-deficient cells. Consistent with this, we observed greater severity and earlier onset of disease in the Th17-mediated EAE model in TRAF4-deficient mice as compared to the littermate controls.
Mechanistically, we found that TRAF4 competes with TRAF6 for binding Act1, resulting in reduced TRAF6-Act1 complex formation and attenuated inflammatory responses. These results are the first to indicate the critical role of TRAF4 in modulating IL-17-mediated signaling and pathogenesis.
IL-17 is a major proinflammatory cytokine that can potently induce and amplify inflammation. Unrestrained, this inflammatory response would eventually cause severe damage to the surrounding tissue. Therefore, sophisticated regulatory mechanisms must be present to restrict this pathogenic pathway. However, little attention has been paid to study negative regulation mechanisms of IL-17 pathway.
We recently described TRAF3 as an IL-17R proximal regulator in critical control of
69 IL-17–mediated inflammatory responses and autoimmune diseases like EAE (Zhu et al., 2010). Binding of TRAF3 to IL-17R interfered with the formation of the activation complex IL-17R–Act1–TRAF6, resulting in suppressed IL-17–mediated signaling and suppressed induction of inflammatory genes (Zhu et al., 2010). In the present study, we observed that TRAF4 also exerts an inhibitory effect on IL-17 mediated inflammatory responses in vitro and in vivo. While IL-17 signaling was substantially enhanced in TRAF4-deficient cells, overexpression of TRAF4 suppressed IL-17-mediated signaling. Furthermore, TRAF4-deficient mice showed aggravated EAE disease development as compared with wild-type mice.
Although both TRAF3 and TRAF4 negatively regulate IL-17-mediated inflammatory responses, the underlying mechanisms are quite different. TRAF3 binds to IL-17R via a putative TB site and hence interferes with the formation of the activation complex IL-17R–Act1–TRAF6 (Zhu et al., 2010). In contrast, TRAF4 competes with TRAF6 for TB sites on Act1. Act1 has two TRAF-binding (TB) sites, both of which are required for TRAF6 binding (Liu et al., 2009). Mutations of these two TB sites abolished TRAF6 binding, leading to the loss of TRAF6-mediated NF- kB activation. Interestingly, we found that these two TB sites are also critical for
TRAF4 binding, implying that TRAF4 and TRAF6 occupy the same binding sites on
Act1. Therefore, the ability for TRAF6 to access these binding sites is enhanced in the TRAF4-deficiency. In the presence of overexpressed TRAF4, the formation of
Act1-TRAF6 complex is greatly reduced, resulting in attenuated downstream signaling and inflammatory responses. Interestingly, one previous study reported that
70 overexpression of TRAF4 could interact and interfere with TRAF6, thereby negatively modulating NF-kB activation in response to LPS (Takeshita et al., 2005).
In contrast to this study we did not detect TRAF6 and TRAF4 association, suggesting that TRAF4 regulates these signaling pathways through differential mechanisms. It is important to note that TRAF4 has also been shown to specifically inhibit NOD2-RIP2-mediated NF-κB activation via binding to NOD-Like Receptors
NOD1 and NOD2 (Marinis et al., 2010). Although TRAF4 exerts its negative regulation via distinct mechanisms in different pathways, it seems that TRAF4 acts as a general inhibitor restricting inflammatory responses.
This study has provided valuable insight into IL-17-mediated pathology and uncovered a previously unknown role for an elusive TRAF-family member. We presented evidence that TRAF4 is a novel negative regulator in IL-17 signaling associated immune responses. TRAF4 interferes with Act1-TRAF6 signaling complex formation by competing for TB sites on Act1. Future studies will need to address whether TRAF4-Act1 interaction is involved in other IL-17-family members. It has been reported that Act1 is the downstream mediator of other IL-17 family members such as IL-17F, IL-25 and IL-17C (Yang et al., 2008;Swaidani et al., 2009;Chang et al.,
2011;Claudio et al., 2009). It will be interesting to see whether TRAF4 plays a similar role in the signaling of these cytokines. It is also important to further examine the role of TRAF4 in other signaling pathways as well as in the pathogenesis of different disease models.
71
Chapter 3:
TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation
Jarod A. Zepp1,2, Ling Wu1,3, Wen Qian1 , Wenjun Ouyang4, Mark Aronica2,5, Serpil Erzurum2,5 and Xiaoxia Li1,2,3
1Department of Immunology, Cleveland Clinic, Cleveland, OH, USA. 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. 3Department of Pathology, Case Western Reserve University 4Department of Immunology, Genentech San Francisco, CA, USA. 5Department of Pathobiology, Cleveland Clinic, Cleveland, OH, USA.
72
I. Abstract
Interleukin-25 (IL-25) can elicit Type-2 immunity by inducing the expression of Th2-
associated cytokines and eosinophilia. While it is known that the IL-25R (IL-17RB)
recruits ACT1 through SEFIR-SEFIR domain interaction, the IL-25R signaling
mechanism remains poorly understood. While screening for additional IL-25
signaling components we found that IL-25-responses were impaired in Traf4 –/–
epithelial cells and T-cells. Administration of IL-25 to the lungs of Traf4 –/– mice
resulted in blunted airway eosinophilia and Th2 cytokine production. Furthermore,
while TRAF4 interacted with the IL-25R in an ACT1-independent manner, ACT1
interaction with the IL-25R was impaired in Traf4 –/– cells. Mechanistically, TRAF4
recruited SMURF2, an E3-ubiquitin ligase, to promote the degradation of an IL-25R-
specific inhibitory molecule, DAZAP2. Silencing of Dazap2 led to increased
ACT1/IL-25R interaction and an enhanced IL-25 response. Moreover, a tyrosine
residue within the IL-25R-SEFIR domain was critical for DAZAP2 interference.
Taken together, this study demonstrates that TRAF4-SMURF2-mediated DAZAP2
degradation is a critical step to initiate IL-25 signaling and airway inflammation.
.
73 I. Introduction
Excessive inflammation in response to otherwise innocuous allergens contributes to the pathology associated with asthma. An important question is how
Th2 cell type immune responses (type-2 responses) are initiated in response to allergen exposure and linked to allergic inflammation. Recent exciting studies have begun to reveal the mechanisms by which the epithelium modulates type 2 responses through the production of a group of epithelial-derived Th2 cell-driving cytokines, including IL-25, IL-33, and TSLP. These epithelial-derived Th2 cell-driving cytokines maintain the balance of host immune homeostasis and defense against various pathogens, whereas dysregulation of these cytokines contributes to excessive type-2 responses and inflammation associated with asthma. In particular, IL-25 induced in airway epithelial cells in response to allergens, has been demonstrated to promote allergic inflammation by directly stimulating Th2-associated cytokine and chemokine production from the airway epithelium as well as from T-cells to exacerbate the pathophysiology of asthma (Angkasekwinai et al., 2007;Kang et al., 2012;Swaidani et al., 2011;Swaidani et al., 2009;Tamachi et al., 2006;Wang et al., 2007).
IL-25 is the most structurally divergent member in the IL-17 cytokine family, exerting distinct physiologic responses (Moseley et al., 2003). While IL-17 cytokines, such as IL-17A, are known to induce neutrophil mobilizing cytokines and chemokines resulting in neutrophil recruitment, IL-25 is the only member demonstrated to initiate type-2-driven inflammation (Fort, et al. 2001;Nakae et al.,
2002;Iwakura et al., 2006). Administration of IL-25 in mice leads to production of the
74 Th2-associated cytokines IL-4, IL-5, IL-9 and IL-13, with eosinophil recruitment and
IgE production (Fort et al., 2001;Kim et al., 2002;Angkasekwinai et al., 2007 ;Claudio et al., 2009;Swaidani et al., 2009;Rickel et al., 2008 ). Elevated levels of IL-25 and its receptor were detected in asthmatic lung tissues, linking their roles in allergic pulmonary inflammation (Wang et al., 2007). In allergic asthma models, mice deficient in IL-25 exhibited reduced cell-infiltrate into the lungs and diminished type-
2 cytokine production (Swaidani et al., 2009;Angkasekwinai et al., 2010;Swaidani et al., 2011;Suzukawa et al., 2012). IL-25 signaling in multiple cell-types, including epithelial cells, type-2 innate lymphoid cells (ILC2s) and T-cells, contribute to IL-25- mediated pathology. Thus, emerging studies have been devoted to target the IL-25 signaling pathway for the development of new strategies for the treatment of asthma and other allergic inflammatory diseases.
The receptors for IL-17A (IL-17RA and IL-17RC for IL-17R) and IL-25 (IL-
17RA and IL-17RB for IL-25R) belong to a common superfamily, defined by a highly conserved SEFIR domain (Similar Expression to FGF genes and IL-17 receptors) in the cytoplasmic region (Novatchkova et al., 2003;Toy et al., 2006;Rickel et al., 2008).
The SEFIR domain facilitates homotypic interactions with other SEFIR domain containing molecules. Work from our group and others have shown that the adaptor molecule known as ACT1 (Activator of NFkB –1, or CIKS), harbors a SEFIR domain, and is a key component in IL-17A and IL-25 signaling (Chang et al.,
2006;Qian et al., 2007;Claudio et al., 2009;Swaidani et al., 2009). We have reported that while mice deficient in Act1 have impaired IL-17-induced pulmonary neutrophil
75 recruitment, Act1 deficiency also abolishes IL-25-induced Th2 cytokines and eosinophil recruitment (Claudio et al., 2009;Swaidani et al., 2009). As a result, Act1–/– mice have reduced allergen-induced pulmonary eosinophilia and inflammatory cytokine production (Swaidani et al. 2009;Swaidani et al., 2011). Upon IL-17 or IL-25 stimulation, ACT1 is recruited to the IL-17R and IL-25R respectively through its
SEFIR domain. Additionally, ACT1 has an E3-ligase U-box domain and TNF- receptor associated factor (TRAF)-binding sites (Liu et al., 2009). These domains allow ACT1 to recognize and ubiquitinate TRAF molecules for subsequent downstream signaling. Specifically, ACT1 mediates K63-linked polyubiquitination of
TRAF6 and TRAF5, which are critical for IL-17-induced NFkB activation and mRNA stabilization of cytokines/chemokines, respectively (Liu et al., 2009;Bulek et al., 2011;Sun et al., 2011). Although there has been much progress in defining the signaling pathways activated by IL-17A, the molecular mechanism of IL-25R-ACT1 induced signal transduction remains elusive.
In this study we screened for TRAF involvement in the IL-25R signaling cascade in primary epithelial cells derived from TRAF -3, -4 and -6 deficient mice.
Using this strategy, we found a striking defect in IL-25 responsiveness in the primary
TRAF4-deficient T cells and epithelial cells, and as well as abolished type-2 responses in Traf4 –/– mice. Mechanistically, TRAF4 mediates the recruitment of the E3-ligase, smad-ubiquitin regulatory factor 2 (SMURF2), to the IL-25R. Moreover, SMURF2 is required for IL-25-induced degradation of the inhibitory molecule deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2). Thus, TRAF4-mediated
76 SMURF2-dependent degradation of DAZAP2 is an essential step in order for IL-25- signaling to commence.
II. Materials and Methods
Mice
TRAF4-deficient (Traf4–/–) C57/BL/6 (B6) mice were generated as described previously (Shiels et al., 2000). SMURF2-deficient (Smurf2–/–) mice were provided by
Dr. Ying Zhang (National Cancer Institute, Bethesda, Maryland) and generated as described in (Tang et al., 2011). IL-17RB-deficient (IL-17rb–/–) mice were obtained from Dr. Wenjun Ouyang (Genentech, San Francisco, California). All experiments used gender and age –matched littermates aged 6-8 weeks. The Institutional Animal
Care and Use Committee of the Cleveland Clinic Foundation approved all animal experiments.
Cell culture, transfection and reagents
Primary kidney epithelial cells were isolated from kidneys taken from the indicated mice aged 10-20 days. Kidneys were minced and incubated in 0.5% Trypsin/EDTA mixture at 37° C for 30 minutes, the supernatant was collected in tubes on ice. This cycle was repeated 3 more times, after which the cells were pelleted and then resuspended in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were grown to confluence at which point the cels were passed for experiments. HEK293 cells were maintained in DMEM plus 10% FBS.
Human bronchial epithelial cell-line (Bet1a) were maintained in LHC9 media (Lonza,
77 Gibco, Life Technologies). Cells were grown on pre-treated dishes and plates with coating media; PureCol (Advanced Biomatix), 1 µg/ml BSA (Gibco), 5 µg/ml
Fibronectin (Cal Biocem) diluted in LHC Basal Media (Gibco) and passed through a
0.22µM filter. Bet1a cells were passed with trypsin-versene solution (Lonza) and trypsin neutralizing solution (Gibco).
For plasmid transfection 1-3 µg of plasmid was transfected using Lipofectamine 2000 reagent (Invitrogen) following the manufacturers’ specifications in Opti-MEM, 24 hours later media was replenished, the following day co-immunoprecipitation was performed. MEF cells stably expressing the IL-25R were described previously (Zhang et al., 2013). Non-targeting shRNA and shRNA targeting murine Dazap2 were purchased from Sigma Aldrich’s mission On-Target shRNA in lentiviral vectors.
Lentivirus was generated by co-transfection of shRNA plasmids with pCl-VSVG and ps-PAX2 (Addgene) using Lipofectamine 2000 into packaging HEK293T cells cultured for 48 hours. MEFs were then transduced with lentiviral particles in the presence of polybrene (5 µg/ml) for 24 hours, following this media was replenished and selected using 1 µ g/ml Puromycin for three days, after which the cells were maintained in 0.5 µg/ml puromycin containing media. Antibodies for immunoblots used are as follows; rabbit anti-ACT1 was described previously in (Qian et al., 2007), goat anti-TRAF4 (N16), mouse anti-Omni tag, rat anti-IL-17RB (TJ5), mouse anti-P-
ERK1/2 and goat anti-ACTIN were from Santa Cruz Biotech. Rabbit anti-P-STAT6, rabbit anti-P-P38, rabbit anti-HA tag, mouse anti-Myc tag, rabbit anti-P-JNK1/2
78 were from Cell Signaling Technologies. Rabbit anti-DAZAP2 was from Abcam.
Mouse anti-HA tag was from Sigma Aldrich. Mouse anti-V5 Tag was from
Invitrogen. Rabbit antibodies against K63- and K48-linked ubiquitination were from
Millipore.
Co-immunoprecipitation and western blotting
For Co-immunoprecipitations, cells were pelleted and lysed in Co-IP Buffer (0.5%
Triton X-100, 20 mM HEPES [pH 7.6], 150 mM NaCl, 12.5 mM b- glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 1mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride [PMSF] and protease inhibitor cocktail tablets
[Roche]) Lysates were incubated on ice for 60 min, and then centrifuged at 13,200 rpm for 10 min and insoluble debris was discarded. The lysates were then incubated overnight with Protein-G sepharose beads conjugated to the indicated antibodies.
The beads were then pelleted and wash 4 times with Co-IP buffer. The precipitates were resolved by SDS-PAGE and subjected to Western blotting with the indicated antibodies.
Ubiquitination assays
Cells were harvested by washing in cold PBS and then lysed in 1% SDS solution. The lysates were sonicated on high setting for 15 s in ice-cold water sonicator
(Diagenode). Then the lysates were boiled for 10 minutes at 100C after which the boiled samples were diluted with Co-IP buffer to 0.1% SDS and the centrifuged at
79 13,200 rpm for 10 min, the pellet was discarded. The supernatant was applied to
Protein-G sepharose beads and antibodies against HA-tag (Sigma Aldrich) and were rotated overnight at 4C. The beads were then pelleted and washed three times with
Co-IP buffer, the precipitates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and subjected to western blotting with the indicated antibodies.
Plasmids
Plasmids for Myc-IL-17RB, Omni-TRAF4, Myc-Smurf2 WT and CG mutant were described previously (Marinis et al., 2011;Tang et al., 2011;Zhang et al., 2013).
Plasmid with full-length Dazap2 was purchased from Genecoepia and subcloned into pCDH-CMV (GFP) lentiviral vector (System BioSciences) using EcoRI and BamHI restriction sites and HA-tag. hsIL-17RB was cloned from cDNA purchased from
Thermo Biosystems, this was further subcloned into lenti- or adenoviral vectors. For adenoviral expression of Il-17rb, the mIL-17rb gene was first subcloned into pENTR/D-TOPO vector. Site-directed mutagenesis was carried out using PFU turbo Taq polymerase (Affymetrix); cDNA sequences were all verified by capillary sequencing. The verified cDNA was then moved to pAd/CMV/V5 DEST adenoviral vector (Invitrogen) according to the manufacturers’ protocol.
Adenovirus production and infection of primary cells
80 Adenovirus production was conducted following the manufacturers’ protocol
(Invitrogen). Briefly, PacI linearized pAd/CMV/V5-IL-17rb Destination vector was transfected into HEK293A producer cells with Lipofectamine 2000 (Invitrogen).
Viral production was typically observed 7-10 days post-transfection, after which cells were pelleted and frozen at -80C. Viral particles were released from pelleted cells by repeated freeze-thaw cycles. The viral stock was then applied at limiting dilutions on confluent cultures of HEK293 cells, plaques were observed within 5 days and enumerated to calculate MOI. Viruses were further titrated in primary kidney epithelial cells, after which western blot for V5-tag was performed. Primary kidney epithelial cells or MEFs were transduced with adenovirus at an MOI of 3 for 24 hours. The next day virus-containing supernatant was removed, cells were washed once with PBS, then fresh media was added and cells were incubated for an additional 24 hours. At this point, cytokine treatment or co-immunoprecipitation studies were performed as indicated. Adenovirus expressing Cre-GFP or null-GFP were purchased from Vector BioLabs and used at a concentration of 1 x 106
PFU/ml.
Intratracheal instillation of IL-25 and IL-17
Instillation of recombinant cytokine was carried out as described previously (Swaidani et al., 2009;Swaidani et al., 2011). Briefly recombinant mouse IL-25 (or IL-17A) without carrier was dissolved in sterile saline, such that 5 µg protein in a volume of 50
µl. Mice were first anesthesized with ketamine/xylazine cocktail, then 50 µ l of
81 recombinant cytokine mix was instilled via sterile syringe and tubing passed through the trachea via the oral cavity (non-surgical). For IL-25 induced airway inflammation, mice were sacrificed and tissue harvested 4 days after the instillation procedure.
Induction of allergic airway inflammation
Mice were sham immunized or OVA-immunized via intra-peritoneal (i.p.) injection with 100 µ g OVA (Grade V, Sigma Aldrich, St. Louis, MO) emulsified with 2 mg
Alum in a total volume of 200 µl in PBS on Day 0 and Day 10. Mice receiving OVA- immunization were subsequently challenged via intra-tracheal administration of 200
µg OVA in 50 µl PBS on day 21, 23 and 25. Mice were sacrificed and BAL fluids and tissue harvested 24 hours after the last challenge.
Brochoalveolar lavage (BAL) and tissue collection
Mice were sacrificed at the times indicated. 600 µl sterile saline was used to obtain
BAL via the trachea using a blunt needle and 1-ml syringe. The BAL fluid was then centrifuged, supernatants were freezed and subsequently used for BAL ELISA, cells were resuspended in PBS and counted via hemocytometer and tyrpan-blue exclusion.
100 µl of the cell suspension was applied to Cytospin cassettes and spun 500xRPM for 5 minutes on Shandon CytoSpin III Cytocentrifuge (Shandon/Thermo Fisher
Scientific). Differential cell counts were determined on cytospin preparations stained using Quik-Diff Giemsa Stain (Thermo Fisher Scientific). Lung tissue was either
82 snap-frozen in liquid nitrogen for further processing for RNA with Trizol or placed in 10% Formalin for paraffin-embedding and H&E and PAS/AB staining.
Statistical analysis and graphing
Raw data-sets were tested for normality by the Kolmogorov-Smirnov test. Data were then analyzed using unpaired t test or the non-parametric Mann-Whitney rank-sum test where appropriate. P-values less than or equal to 0.05 were considered statistically significant. All statistics and graphical representations were conducted with Prism 5.0 software for Mac (Graphpad).
Acknowledgements
This work was supported by NIH grants R01-NS071996 (NINDS) and P01-
HL103453 (NHLBI) to X.L. J.Z. is a Ph.D. student in the Molecular Medicine Ph.D. program of Cleveland Clinic and Case Western Reserve University, funded in part, by the Med into Grad initiative of the Howard Hughes Medical Institute and
T32GM088088 from NIH.
83 III. Results
Differential requirements for TRAF proteins in the IL-25 response
Previous studies have shown that ACT1 is the key adaptor molecule recruited to the IL-17 and IL-25 receptors following ligand stimulation (Claudio et al.,
2009;Swaidani et al., 2009). ACT1 functions as an E3-ubiquitin ligase and interacts with specific TRAF proteins to direct downstream signaling pathways. Given the differential responses elicited by IL-17 and IL-25 in vivo, we sought to test how specific TRAF proteins may participate in the IL-25 response. As reported previously, we observed a marked reduction in IL-17- and IL-25-induced Cxcl1 gene expression and loss of ligand-induced IkBa phosphorylation in the TRAF6-deficient cells, confirming the critical role of TRAF6 for IL-17- and IL-25-mediated NFkB- dependent gene induction (Fig. 3-1A and data not shown) (Schwandner et al.,
2000;Hartupee et al., 2009). Since TRAF3 and TRAF4 have been reported as negative regulators for IL-17-induced NFkB activation, we next aimed to determine the roles of these two TRAFs in IL-25 signaling. We isolated Traf3flox/flox kidney cells and infected with an adenovirus expressing Cre recombinase to mediate Traf3 deletion
(data not shown). There was no significant difference in IL-25-induced expression of
Cxcl1 or IL-25 in the TRAF3-deficient cells compared to that in wild-type cells (Fig.
3-1B-C), whereas Traf3 –/– cells were indeed hyper-responsive to IL-17 (Fig. 3-1B)
(Zhu et al., 2010). These results indicate that TRAF3 is dispensable for the IL-25 response. Importantly, whereas the primary kidney epithelial cells derived from the
Traf4 –/– mice were also hyper-responsive to IL-17-induced Cxcl1 expression, IL-25-
84 induced expression of Cxcl1 or IL-25 was abolished in TRAF4-deficient cells (Fig. 3-
1D-E). These data suggest that TRAF4 is differentially required for IL-17- and IL-25- mediated signaling (Zepp et al., 2012). In summary, the screening of TRAF proteins revealed an unexpected essential role for TRAF4 in mediating an IL-25 response.
85
Figure 3-1. Screening for TRAF involvement in IL-25R-dependent response. Primary cells were transduced with adenovirus encoding IL-25R for 48 hours. Cells were treated with IL-25 or IL-17A for the indicated times, RNA was isolated and real-time qPCR performed. (a) Cxcl1 gene expression in MEFs from Traf6 –/– or WT mice. (b and c)
86 Kidney epithelial cells (KEC) were isolated from Traf3 f/f mice. Cells were transduced with –AdCre and were then stimulated as indicated and Cxcl1 (b) or Il-25 (c) was measured. (d and e) KEC isolated from WT or Traf4 –/– mice were infected and treated same as in (a-c). All data are representative results from at least 2 or 4- independent experiments; gene expression data are normalized to b-actin, error bars represent mean ± SEM.
TRAF4 is required for IL-25-dependent airway inflammation
IL-25 was first reported as a cytokine capable of initiating Type-2 associated pathology and cytokine production (Angkasekwinai et al., 2007). IL-25 administration into the airways of mice induces the production of IL-4, IL-5, IL-13 and subsequent eosinophilia (Rickel et al., 2008;Claudio et al., 2009;Swaidani et al.,
2009). Thus, in order to corroborate our in vitro findings we tested whether TRAF4 is critical for IL-25 responses in vivo. We administered recombinant IL-25 via intra- tracheal injection to Traf4 –/– and WT littermates. Although we observed significant cell accumulation in the broncho-aveolar lavage (BAL) fluid of control mice treated with IL-25, this effect was abolished in the Traf4 –/– mice (Fig. 3-2A). The BAL differential cell counts revealed that IL-25-induced eosinophilia was greatly reduced in the Traf4 –/– mice compared to that in the littermate control mice (Fig. 3-2B).
Further examination of the lung histology revealed substantial IL-25-induced accumulation of immune cells and mucus production in the small airways of control mice, which was absent in the Traf4 –/– mice (Fig. 3-2C). Furthermore, IL-25- induced expression of type-2 cytokines, including Il-4, Il-5, Il-13, Il-9, Il-25 and Ccl11
(eotaxin) was greatly reduced in the Traf4 –/– lung tissue compared to that of the wild-
87 type mice (Fig. 3-2D). Moreover, IL-5 and IL-13 recovered from the BAL fluid were also significantly reduced in the Traf4 –/– mice compared to that in the control mice
(Fig. 3-2E). Taken together, these observations indicate the critical role of TRAF4 in the IL-25-induced Type-2 response in vivo. Notably, we also tested the impact of
TRAF4 deficiency on IL-17 response in vivo and found that Traf4 –/– mice exhibited increased IL-17-induced cell accumulation and target gene expression in the lungs
(Fig. 3-3). These data further support the specificity of TRAF4 for the IL-25 signaling pathway.
Figure 3-2. TRAF4 regulates IL-25 responses in vivo. WT and Traf4 –/– mice were injected with IL-25 (5µg per mouse) via intra-tracheal administration. After 4 days (a) Total cellularity recovered from bronchial-aveolar lavage (BAL) fluid was enumerated. (b) Cell differential from BAL determined by Wright-Geimsa stain.
88
Figure 3-2. TRAF4 regulates IL-25 responses in vivo. (c) IL-25-treated lungs were fixed in formalin and stained with H&E and PAS/AB, shown are representative images.
89
Figure 3-2. TRAF4 regulates IL-25 responses in vivo. (d) Lung tissue RNA profile for the indicated genes from treated and untreated mice as determined by RT-qPCR normalized to b-actin. (e) ELISA for IL-5 and IL-13 from the BAL fluid of untreated and treated mice. Results shown are representative data with n=5 mice per experimental group from 2 independently performed experiments, error bars represent ± SEM, * indicates p<0.05, ** p<0.01, *** p<0.001.
90
Figure 3-3. Enhanced IL-17A-induced airway inflammation in TRAF4-deficient mice. (a) WT and Traf4–/– mice were administered IL-17A (5 µg/mouse) via intratracheal route, 24 hours later, BAL fluid was collected and total cellularity determined. (b) Cell differential from BAL fluid from (a). (c) Lung tissue RNA was extracted and RT- qPCR was performed for the indicated gene normalized to b-actin. Data is representative from two independent experiments with n=4-5 mice per group, error bars represent mean ± SEM, * indicates p<0.05, ** p<0.01.
91 The OVA-sensitization and challenge model has been used extensively to recapitulate allergen-induced airway inflammation in mice. Moreover, several studies have implicated IL-25 as a critical initiator of many downstream consequences following allergen exposure (Angkasekwinai et al., 2007;Swaidani et al.,
2009;Angkasekwinai et al., 2010;Swaidani et al., 2011;Suzukawa et al., 2012). Since we found that TRAF4 is an essential signaling component of the IL-25-induced Type-2 response, we then examined the impact of TRAF4 deficiency on OVA-induced airway inflammation. We observed that OVA-induced BAL cellularity was substantially reduced in Traf4 –/– mice compared to that in wild-type control mice
(Fig. 3-4A). Specifically, eosinophil and lymphocyte recruitment ware markedly decreased in the absence of TRAF4 (Fig. 3-4B). Importantly, the marked decrease in inflammation in Traf4 –/– mice was similar to the defects observed in Il-17rb –/– mice
(data not shown). Moreover, the Traf4 –/– mice exhibited a reduced gene expression profile for type 2 cytokines (Il-4, Il-9, Il-13, Il-25) as well as reduced eotaxin-1 and muc5ac (Fig. 3-4C). Together, these data implicate TRAF4 as a crucial molecule in allergen-induced IL-25-dependent airway inflammation.
92
Figure 3-4. TRAF4 mediates allergic airway inflammation. WT and Traf4–/– mice were immunized with OVA emulsified in Alum on Day 0 and Day 10 followed by 3 subsequent intratracheal challenges with OVA on Day 21-23. (a) BAL wash was performed and total cellularity and cell differential was determined.
93
Figure 3-4. TRAF4 mediates allergic airway inflammation. (b) Lung tissue from OVA- treated mice were fixed and H&E stained, representative sections are shown.
94
Figure 3-4. TRAF4 mediates allergic airway inflammation. (c) Lung tissue RNA was extracted and RT-qPCR was performed for the indicated genes normalized to b-actin. Data is representative from two independent experiments with n=4-5 mice per group, error bars represent mean ± SEM, * indicates p<0.05, *** p<0.001.
TRAF4 is required for IL-25 response in T-cells and airway epithelial cells
Using cell-type specific ACT1-deficient mice, we have previously reported that
T-cell- and epithelial-derived Act1 expression is critical for the IL-25 response in vivo
(Swaidani et al., 2009;Swaidani et al., 2011). Thus we tested the role of TRAF4 in IL-
25-mediated responses in these cell-types. Whereas Traf4 –/– Th2 cells (polarized by
IL-4) exhibited no difference in cytokine production compared to that in littermate controls, Traf4 –/– Th25 cells (polarized by IL-25) showed substantially reduced IL-5 and IL-13 production and expression of Il-4 and Gata-3 compared to that in wild-type
Th25 cells (Fig. 3-5A-B). As controls, we show that TRAF4 deficiency in T cells had
95 no impact on Il-25R (Il-17rb), Il-17ra and Ifnγ expression or cell proliferation (Fig. 3-
5B and data not shown). To assess the role of TRAF4 in mediating the IL-25 response in airway epithelial cells, we knocked down Traf4 in a human airway epithelial cell-line, Bet1A (Fig. 3-5C). IL-25-induced expression of Il-13, Ccl11
(eotaxin) and Ccl5 (rantes) was greatly reduced in the TRAF4-silenced (siTraf4) cells compared to the cells transfected with non-targeting scrambled siRNA (Fig. 3-5D).
As a control, we found that IL-1β-induced CXCL1 expression was unaffected by
TRAF4-silencing compared to the control cells (data not shown). These data indicate that TRAF4 is specifically required for IL-25 responses in T-cells as well as in human airway epithelial cells.
96
Figure 3-5. Cell-intrinsic IL-25 responses are TRAF4-dependent. Naïve CD4-positive T- cells were isolated from WT and Traf4 –/– mice and subsequently activated with plate-bound CD3/CD28 and in the indicated polarizing conditions. (a) ELISA for IL-5 and IL-13 performed from supernatants of activated T-cells. (b) RNA was isolated from activated T-cells in Th0+IL-25 conditions and RT-qPCR for the indicated genes normalized to β-actin.
97
Figure 3-5. Cell-intrinsic IL-25 responses are TRAF4-dependent. (c) Immunoblot from human airway epithelial cell-line (Bet1a) transfected with siRNA against human Traf4 or scrambled (scr) (100 nM each). (d) Bet1a cells transfected with siRNA against Traf4 or scr control were treated with hIL-125 (100 ng/ml) for the indicated times, RNA was isolated and RT-qPCR was performed for the indicated genes normalized to gapdh.
98
Consistent with the decreased IL-25-dependent T-cell cytokine response, IL-
25-induced phosphorylation of ERK1/2 and P38 were abolished in the Traf4 –/– T cells as compared to that in wild-type T cells (Fig. 3-5E).
Figure 3-5. Cell-intrinsic IL-25 responses are TRAF4-dependent. (e) Activated T-cells in Th2 polarizing conditions from WT and Traf4 –/– mice were treated with IL-25 (100 ng/ml) for the indicated times, lysates were subjected to SDS-PAGE followed by immunoblotting for the indicated proteins.
99
One important question is how TRAF4 impacts on IL-25 signaling. We performed co-immunoprecipitation with the IL-25R to assess whether TRAF4 is recruited to the receptor complex. Although there was constitutive interaction of
TRAF4 with the IL-25R, IL-25 stimulation enhanced the recruitment of TRAF4 to
IL-25R (Fig. 3-5F). Surprisingly, we observed a substantial defect in the recruitment of ACT1 to the IL-25R in Traf4 –/– cells, implicating a critical role of TRAF4 in the recruitment of ACT1 to IL-25R (Fig. 3-5F). On the other hand, TRAF4 was still able to interact with IL-25R in Act1 –/– cells, indicating that the recruitment of TRAF4 to
IL-25R is primarily ACT1-independent (Fig. 3-5G).
100
Figure 3-5. Cell-intrinsic IL-25 responses are TRAF4-dependent. (f and g) WT and Traf4 – /– or (g) Act1 –/– , KECs were isolated and infected with Ad-V5-Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5.
101 Mutations within the putative TRAF binding site (334-341: VYPSEICF, to
S337A, E338A and I339A) in IL-25R markedly impaired its interaction with TRAF4
(Fig. 3H) . Collectively, these results suggest that IL-25R/TRAF4 interaction may be a pre-requisite for subsequent ACT1 recruitment.
Figure 3-5. Cell-intrinsic IL-25 responses are TRAF4-dependent. (h) HEK293 cells were transfected with the indicated vectors. After 48 hours the cell lysate was prepared followed by co-immunoprecipitation for 24 hours using antibodies against MYC-tag IL-25R. (f-h) Co-immunoprecipitate (Wang et al.) and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. All data are representative of at least 3 independently performed experiments. Error bars represent mean ± SEM, * indicates p<0.05, ** p<0.01, *** p<0.001.
102 TRAF4 and SMURF2 cooperate to mediate the IL-25 response
Since it has been well defined that ACT1 is recruited to IL-25R through
SEFIR-SEFIR domain interaction, it is critical to identify a possible mechanism that could explain the TRAF4-dependent recruitment of ACT1 to the IL-25R (Swaidani et al., 2009;Liu et al., 2011;Zhang et al., 2013). One possibility is that TRAF4 is recruited to IL-25R to remove a pre-bound inhibitory molecule of the receptor to permit the recruitment of ACT1. Previous yeast hybrid screening with the intracellular portion of IL-25R as bait identified a novel binding partner known as deleted in azospermia
(DAZ)-associated protein 2 (DAZAP2) (Popova et al., 2012). In that study, SMURF2
(SMAD-ubiquitin regulatory factor) was identified as the E3-ligase capable of promoting DAZAP2 degradation. However, the functional importance of DAZAP2 or SMURF2 in IL-25 signaling has not been established. Notably, several reports have shown that TRAF4 interacts with SMURF2 in several contexts, including metastatic breast cancer and in the TGFb signaling pathway (Li et al., 2010;Wang et al., 2013;Zhang et al., 2013). Our results here show that TRAF4 is required for IL-25- induced recruitment of ACT1 to the IL-25R. Thus we hypothesize that if DAZAP2 inhibits the recruitment of ACT1 to the IL-25R then TRAF4 may mediate the recruitment SMURF2 to the IL-25R complex in order to degrade DAZAP2 and remove its inhibitory activity.
To test this hypothesis, we first assessed the impact of SMURF2 in IL-
25 signaling. Consistent with the previous reports, we indeed observed the TRAF4-
SMURF2 interaction (Fig. 3-6A) (Li et al., 2010;Wang et al., 2013). Moreover,
103 SMURF2 interacts with the IL-25R in primary cells and the binding of SMURF2 to the IL-25R was substantially enhanced when increasing amounts of TRAF4 (Fig. 3-
6B-C), suggesting that TRAF4 facilitates the recruitment of SMURF2 to IL-25R.
Consistent with our observation in the Traf4 –/– cells, the ACT1/IL-25R interaction was substantially diminished in Smurf2 –/– cells (Fig. 3-6C). These data suggest that
TRAF4 can facilitate SMURF2 recruitment to the IL-25R complex, which is required for subsequent recruitment of ACT1.
104
Figure 3-6. SMURF2 is a positive mediator of the IL-25-response. (a and b) HEK293 cells were transfected with the indicated vectors. After 48 hours the cell lysate was prepared followed by co-immunoprecipitation for 24 hours using antibodies against MYC-tag SMURF2 (a) or V5-tag IL-25R (b). (c)WT and Smurf2 –/– KECs were isolated and infected with Ad-V5-Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. The co-immunoprecipitate (Wang et al.) and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (a-c) Data are representative results of at least 2 independently performed experiments.
105 To further substantiate the involvement of SMURF2 in IL-25 signaling, we tested the impact of SMURF2 deficiency on IL-25 responses in vivo. We found that IL-25-induced BAL cellularity was significantly reduced in Smurf2–/– mice compared to WT mice (Fig. 4D). Consistent with this, SMURF2 deficiency reduced
IL-25-induced eosinophilia (Fig. 4E). Similarly, inflammation and mucus production were also diminished in the lung tissue of Smurf2–/– mice (Fig. 4F). Moreover, the known IL-25 target genes, Il-25 and Il-13, were also both significantly reduced in the lung tissue and in the BAL fluid of Smurf2–/– mice (Fig. 4G-I). Together, these results support the hypothesis that TRAF4 and SMURF2 positively facilitate the IL-25 response.
106
Figure 3-6. SMURF2 is a positive mediator of the IL-25-response. (d) WT and Smurf2 –/– mice were injected with IL-25 by intra-tracheal route. After 4 days BAL wash was performed and total cellularity (d) and eosinophil in the BAL (e) were enumerated. (f) Lungs were sectioned and stained as indicated. Representative images from treated mice are shown.
107
Figure 3-6. SMURF2 is a positive mediator of the IL-25-response. (g-h) RNA was prepared from total lung tissue and RT-qPCR performed for Il-25 and Il-13 normalized to β- actin. (i) IL-13 ELISA from BAL fluid of treated mice. Data presented are from a representative experiment with n=4 mice per experimental group, thses experiments were repeated 2 times with similar results. Error bars represent mean ± SEM, * indicates p<0.05, ** p<0.01.
108 TRAF4 and SMURF2 are required for IL-25-induced degradation of the inhibitory molecule DAZAP2
Considering the previous report about the connection between
SMURF2 and DAZAP2, we next examined the impact of DAZAP2 on the IL-25 response. Primary mouse T-cells were transduced with control shRNA or lentiviral shRNA targeting Dazap2 (Fig. 3-7A). We then polarized the shDazap2 or scrambled shRNA control T-cells with IL-25 and then measured cytokine expression. We observed substantially more IL-25-induced Il-5 and Il-13 gene expression as well as
IL-5 protein production in the T-cells with shDazap2 compared to the control (Fig.
3-7B-C).
109
Figure 3-7. DAZAP2 negatively impacts IL-25 responses. (a) Primary murine T-cells were transduced with lentivirus expressing shRNA targeting Dazap2 or non-targeting scrambled. RNA was isolated and RT-qPCR was performed 5 days after initial transduction and normalized to actin. (b) T-cells from (a) were activated by plate bound CD3/CD28 in the indicated conditions. RNA was isolated and RT-qPCR for the indicated genes was performed and expression normalized to b-actin. (d) IL-5 ELISA from T-cell supernatant in (b-c).
110 To further determine the impact of DAZAP2 on IL-25-induced signaling, we also generated a Dazap2-knockdown MEF cell line (shDazap2) (Fig. 3-7E). Compared to the control cell-line (scr), the shDazap2 cells exhibited enhanced IL-25-induced activation of ACT1, JNK1/2, ERK1/2 and P38 (Fig. 3-7F). Importantly, IL-17 induced comparable activation of ACT1, JNK1/2, ERK1/2 and P38 in the scr and shDazap2 cells (Fig. 3-7G), indicating that DAZAP2 specifically inhibits IL-25R- mediated signaling.
111
Figure 3-7. DAZAP2 negatively impacts IL-25 responses. (e) IL-25R expressing MEF cell line were transduced with lentiviral expressed shRNA targeting Dazap2, RNA was prepared and RT-qPCR performed and normalized to actin. (f and g) MEFs from (e) were treated with IL-25 (f) or IL-17A (g) for the indicated times, lysate was prepared and immunoblotted for the indicated proteins.
112 Moreover, we observed a significant increase in the interaction of ACT1 with the IL-
25R in the shDazap2 cells compared to the control cells (scr) (Fig. 3-7H). These observations suggest that DAZAP2 hinders the recruitment of ACT1 to the IL-25R, resulting in the inhibition of IL-25 signaling.
Figure 3-7. DAZAP2 negatively impacts IL-25 responses. (h) MEF cells were treated with IL-25, lysate was prepared and co-immunoprecipitated with antibodies against ACT1, then immunoblotted for the indicated proteins. Data are representative results from 2-3 independently performed experiments. Error bars represent mean ± SEM, * indicates p<0.05, ** p<0.01.
Thus far, our results indicate that TRAF4 and SMURF2 are required for
ACT1 recruitment to the IL-25R. In contrast to this, DAZAP2 hinders the
ACT1/receptor interaction and inhibits downstream IL-25 signaling. An important point to address is how IL-25-stimulation can overcome DAZAP2 inhibition, and whether TRAF4 and SMURF2 participate in this process. We examined the impact of
IL-25 stimulation on endogenous DAZAP2 in polarized Th2 from WT, Traf4 –/– or
Smurf2 –/– mice. DAZAP2 levels were substantially reduced within 15 minutes of IL-
25 treatment and this reduction was dependent on TRAF4 and SMURF2 (Fig. 3-8A).
113
Figure 3-8. IL-25 stimulation promotes DAZAP2 degradation. (a) Primary CD4+ TH2 cells derived from the indicated mice were treated with IL-25, cell lysate was prepared and subjected to SDS-PAGE and immunoblotting for DAZAP2. Representative blots from at least 2 or 3 independently performed experiments are shown, ratio is relative to GAPDH loading control.
It was previously reported that SMURF2 is the E3-ligase capable of promoting proteasome-dependent DAZAP2 degradation (Popova et al., 2012). Interestingly, we found IL-25 stimulation induced K48-, but not K63-linked polyubiquitintation of
DAZAP2 within 5 minutes of IL-25 stimulation in MEFs (Fig. 3-8B). Moreover,
MG132 blocked IL-25-induced DAZAP2 degradation, resulting in accumulation of
K48-linked polyubiquitintation of DAZAP2 (Fig. 3-8B). These results suggest that
IL-25 stimulation can lead to DAZAP2 ubiquitination and degradation, thereby relieving DAZAP2-mediated inhibition. Consistent with the results in MEFs and Th2 cells, IL-25 also induced DAZAP2 degradation in primary kidney epithelial cells within 2-5 minutes of stimulation, which was abolished in the Traf4 –/– cells (Fig. 3-
8C).
114
Figure 3-8. IL-25 stimulation promotes DAZAP2 degradation. (b) IL-25R expressing MEF cells with stable expression of HA-tagged DAZAP2 were treated with IL-25 for the indicated timepoints, cell lysates were prepared and co-immunoprecipitated with antibodies against HA-tag (top), then immunoblotted for the indicated proteins and endogenous Ub. (b, bottom) quantification of whole cell lysate DAZAP2/GAPDH ratio. MG132 was incubated for 2 hrs prior to lysate preparation where indicated.
115
Figure 3-8. IL-25 stimulation promotes DAZAP2 degradation. (c) Primary KECs derived from WT or Traf4 –/– were transduced with viruses encoding HA-Dazap2 and V5-IL- 25R after which, cells were stimulated with IL-25 for the indicated times, cell lysate was prepared and separated by SDS-PAGE followed by immunoblotting for HA-tag.
SMURF2 was previously implicated as the E3 ligase mediating DAZAP2 degradation.
We indeed found that wild-type SMURF2, but not an E3-ligase defective mutant
SMURF2-CG, was able to promote DAZAP2 degradation, which was also blocked by MG132 (Fig. 3-8D). Co-expression of WT SMURF2 but not the E3-defective mutant could promote poly-ubiquitination of DAZAP2 (Fig. 3-8E). Moreover, this ubiquitination was K48-linked, as a K48R mutant Ub failed to assemble on DAZAP2
(Fig. 3-8F). These results clearly suggest that TRAF4 and SMURF2 mediate IL-25 signaling by promoting IL-25-induced K48-linked polyubiquitintation of DAZAP2 and consequent degradation, thereby alleviating the inhibition of ACT1 recruitment imposed upon by DAZAP2.
116
Figure 3-8. IL-25 stimulation promotes DAZAP2 degradation. (d and e) HEK293 cells were transfected with the indicated vectors, lysate was prepared and then separated by SDS-PAGE. MG132 was incubated for 2 hrs prior to lysate preparation where indicated. All blots shown are from one representative experiment performed 2 or 3 times.
117
IL-25R recruits DAZAP2 through p-Tyr 355 residing within the SEFIR domain
We next further investigated the molecular basis for DAZAP2-mediated inhibition on ACT1 recruitment to the IL-25R. DAZAP2 contains three putative
SH2 domains (Fig. 3-9A) (Shi et al., 2007). It is well known that SH2 domains facilitate protein-protein interaction through the recognition of phosphorylated tyrosine residues. Interestingly, we indeed noted multiple tyrosine residues (six) in the intracellular portion of the IL-25 receptor (Fig. 3-9A). Mass spectrometry data deposited in the post-translational modification database Phospho-site Plus, indicates several of the tyrosine residues are phosphorylated, including 306, 444 and 463. It is important to note that five of these tyrosine residues reside within the SEFIR domain, which is the requisite domain for ACT1-SEFIR interaction. Thus we hypothesized that the IL-25R may harbor phosphorylated tyrosines (in the SEFIR domain) that are essential for the recognition and binding of the SH2 domains in
DAZAP2, hindering the recruitment of ACT1 to the SEFIR domain of the receptor.
118
Figure 3-9. IL-25R contains tyrosine residues that modulate its function. (a) Schematic diagram showing tyrosine residues on the intracellular domain of IL-17RB and previously mapped DAZAP2 binding region, (right) schematic of SH2 domains on DAZAP2.
To determine the importance of SH2 domains in DAZAP, we generated
SH2-domain deletion mutants of DAZAP2. Deletion of any one of the SH2 domains impaired DAZAP2’s interaction with the IL-25R (Fig. 3-9B), which suggests the possible recognition of phosphorylated tyrosine residues on the IL-25R. We then generated a compound tyrosine mutant in which all six of the tyrosine residues in the
IL-25R were mutated to phenylalanine (All-Tyr mutant). We observed a dramatic shift in the mobility of the All-Tyr mutant compared to the wild-type IL-25R when they were re-expressed in IL-25R-deficient kidney epithelial cells (Fig. 3-9C). These results suggest that the IL-25R is highly modified and that the tyrosine residues on the intracellular portion contribute to this modification. Using anti-p-Tyr antibody,
119 we indeed detected tyrosine phosphorylation in IL-25R, which was further induced by IL-25 stimulation (Fig. 3-9D).
Figure 3-9. IL-25R contains tyrosine residues that modulate its function. (b) HEK293 cells were transfected with the indicated vectors, cells were pelleted and lysates were prepared and co-immunoprecipitated with antibodies against HA-tag, following this lysates were subjected to SDS-PAGE and immunoblotting. (c) WT KEC were transduced with Ad-IL-17RB and treated with IL-25 for the indicated times, cells were pelleted and lysates were prepared and co-iummunoprecipitated with antibody against P-Tyr, after which lysates were subjected to SDS-PAGE and immunoblotting. (d) WT KEC were transduced with Ad-IL-17rb (WT) or Ad-IL-17rb with all intracellular tyrosines mutated to phenylalanine (All-Tyr) for 48 hours followed by SDS-PAGE and immunoblotting.
120 To test whether these tyrosines are required for DAZAP2 recruitment to the receptor, we generated site-specific mutants and co-expressed with DAZAP2.
While WT IL-25R, Y335F and Y440F retained the interaction with DAZAP2, Y355F lost the interaction with DAZAP2 (Fig. 3-9E). In order to study the regulation and function of the Y355F mutant, we re-introduced this variant and WT IL-25R into IL-
17rb –/– kidney epithelial cells. Interestingly, Y355F showed much reduced basal tyrosine phosphorylation compared to wild-type IL-25R, suggesting that phosphorylation at this site probably takes place in the absence of IL-25 stimulation
(Fig. 3-9F). Furthermore, while wild-type IL-25R could restore IL-25 response in IL-
17rb –/– cells, we actually saw a markedly enhanced response to IL-25 in the Y355F restored kidney epithelial cells (Fig. 3-9G). These results indicate that the mutant IL-
25R lacking the tyrosine necessary for DAZAP2 recruitment is capable of transducing a stronger IL-25 response, confirming the inhibitory role of DAZAP2 on
IL-25 signaling. Since this tyrosine residue resides in the SEFIR domain, it is logical to propose that the recruitment of DAZAP2 to this site probably would directly interfere with the ACT1/IL-25R interface (mediated by the SEFIR domain). We indeed observed that DAZAP2 could no longer inhibit ACT1’s interaction with the mutant IL-25R Y355F compared to the wild-type IL-25R (Fig. 3-9H).
121
Figure 3-9. IL-25R contains tyrosine residues that modulate its function. (e) HEK293 cells were transfected with the indicated vectors followed by co-immunoprecipitation against HA-tag and immunoblotting. (f) Il-17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the Y355F mutant, 48 hours later, cell lysates were prepared and co-immunoprecipitated with antibody against P-Tyr, after which immunoblotting was performed. (g) Il-17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the indicated single Y to F mutants, (top) is immunoblot for the transduced receptors (bottom) KEC were treated for the indicated time with IL-25, RNA was isolated and RT-qPCR performed and normalized to b-actin. (h) HEK293 cells were transfected with the indicated vectors, cells were pelleted and lysates were prepared and co-immunoprecipitated with antibodies against Myc-tag, following this lysates were subjected to SDS-PAGE and immunoblotting.
122
IL-25R Y355F mutant mediates IL-25 response in the absence of TRAF4
Since TRAF4 is required for IL-25-induced DAZAP2 degradation, one would predict that the mutant IL-25R Y355F (lacking the tyrosine necessary for
DAZAP2 recruitment) would bypass the need for TRAF4 to mediate the IL-25 response. To test this, we transduced wild-type and IL-25R Y355A mutant into wild- type and Traf4 –/– kidney epithelial cells, followed by treatment with IL-25.
Importantly, we found that while the wild-type IL-25R still requires TRAF4 to respond to IL-25, IL-25R Y355F mutant was able propagate an IL-25 response in the absence of TRAF4 (Fig. 7I). Overall these results indicate that post-translational modification of the IL-25R determines DAZAP2 recognition and thus TRAF4 involvement. Collectively, the TRAF4-SMURF2 regulatory axis is essential for
DAZAP2 degradation, and most likely prepares the receptor to recruit the key adaptor molecule ACT1, an essential permissive event to initiate IL-25 signaling (Fig.
7J).
123 i. j. .
Figure 3-9. IL-25R contains tyrosine residues that modulate its function. (i) Traf4 –/– KECs were transduced with adenovirus encoding for IL-17rb WT or Y355F mutant. After 48 hours cells were treated with IL-25 (100 ng/ml) for the indicated times, RNA was isolated and RT-qPCR performed and normalized to b-actin. Representative data of 2 or 3 independently performed experiments are shown, error bars represent mean ± SEM. (j) Working model for IL-25 signaling pathway, (left) the IL-25R in unstimulated cells is suppressed by DAZAP2 binding to tyrosine-355. (right) Upon ligand stimulation, TRAF4 is recruited to the Traf-binding site (TB) and recruits SMURF2 to the receptor complex. SMURF2 polyubiquitinates DAZAP2 leading to its degradation. This results in ACT1 recruitment and subsequent activation of IL-25 responsive genes.
124 Discussion
In this study we identified a novel role for TRAF4 in mediating the IL-25 response. TRAF4 deficiency impaired the IL-25-induced Th2 response in vivo, in cultured T-cells and epithelial cells. While TRAF4 interacted with the IL-25R in an
ACT1-independent manner, ACT1 interaction with the IL-25R was impaired in Traf4
–/– cells. We demonstrated that the positive activity of TRAF4 was to recruit
SMURF2, an E3-ubiquitin ligase, to promote the degradation of an IL-25R-specific inhibitory molecule, DAZAP2. Silencing of Dazap2 led to increased ACT1/IL-25R interaction and an enhanced IL-25 response. Moreover, a tyrosine residue within the
IL-25R-SEFIR domain was critical for DAZAP2 interference. Taken together, this study demonstrates that TRAF4-SMURF2-mediated DAZAP2 degradation is a critical step for the recruitment of ACT1 to IL-25R to initiate IL-25 signaling and airway inflammation.
The identification of TRAF4 as a critical component of the IL-25 response was unexpected. We reported that in the IL-17A signaling cascade, TRAF4 is a negative regulator of NFkB activation and IL-17-associated disease pathology
(Zepp et al., 2012). Under the IL-17A pathway, TRAF4 binds to ACT1 and occupies the TRAF-Binding-Domains (TBDs) on ACT1 thereby blocking TRAF6. However, while TRAF4-IL-25R interaction is dependent on a putative TRAF binding site (334-
341: VYPSEICF) in IL-25R, TRAF4 is required for ACT1 recruitment to the IL-25R, suggesting that TRAF4 may directly interact with the IL-25R preceding ACT1.
Indeed, TRAF4 deficiency blunted IL-25-responses in primary cells and in IL-25-
125 driven models of airway inflammation. The mechanism for how TRAF4 facilitates the recruitment of ACT1 to the IL-25R was indirect. Rather, an inhibitory molecule,
DAZAP2, hinders the recruitment of ACT1 to the IL-25R. IL-25 stimulation elicits
TRAF4 to the receptor complex to remove this inhibitory effect.
DAZAP2 is highly conserved among mammals and its secondary structure features several SH2 and SH3 domains (Shi et al., 2004; Shi et al., 2007). We identified tyrosine residue 355 of the IL-25R serves as the docking site for the SH2 domain of DAZAP2. It is important to note that both the TRAF binding site and tyrosine-355 reside in the SEFIR domain of IL-25R, which is known to interact with the SEFIR domain of ACT1 (Liu et al., 2011;Zhang et al., 2013). Since silencing
Dazap2 increased ACT1 recruitment to the IL-25R and enhanced IL-25 signaling, it is likely that DAZAP2’s binding to the p-Tyr in the SEFIR domain creates a steric interference for the SEFIR-SEFIR interaction between ACT1 and IL-25R. Our data suggests that binding of TRAF4 to the N-terminal end of the SEFIR (SEFIR: 329-
477 versus TRAF binding site: 334-341) is cooperative for SEFIR-SEFIR interaction.
Furthermore, since ACT1 also contains the TRAF binding site, TRAF4 may be displaced from the IL-25R and turn into an ACT1-TRAF4 complex after the engagement of SEFIR-SEFIR domains. The IL-25 response in Traf4 –/– cells was restored with IL-25R-Y355F, suggesting that TRAF4 may be dispensable beyond its effects on DAZAP2. However, genome wide gene array studies will be helpful to assess whether TRAF4 is cooperative with ACT1 following removal of DAZAP2.
126 Importantly, we found that TRAF4 participates in IL-25 signaling by partnering with another E3 ligase SMURF2 to mediate K48-linked polyubiquitination and consequent degradation of DAZAP2, thereby allowing ACT1 recruitment to the
IL-25R. Since both TRAF4 and SMURF2 are required for IL-25-induced ACT1 recruitment and IL-25 signaling, their roles in this IL-25-dependent process must be non-redundant. Our results demonstrated that TRAF4 is directly recruited to IL-25R in response to ligand stimulation. TRAF4 interaction with the IL-25R facilitates
SMURF2 recruitment, which in turn, interacts with and degrades DAZAP2.
SMURF2 was previously identified as the E3-ligase capable of inducing proteasome- dependent DAZAP2 degradation (Popova et al., 2012). It is important to note that neither IL-25-induced modifications nor degradation of TRAF4 or SMURF2 were ever observed in our study (unpublished observations). Therefore, for the IL-25R pathway we conclude that the main activities of TRAF4 and SMURF2 are conducted in a cooperative manner to promote DAZAP2 degradation.
It is exciting to note that this is the first example of the IL-25R, or any
IL-17R, being modified at the post-translational level. Moreover, we detected tyrosine phosphorylation of IL-25R in the absence of IL-25 stimulation. Thus, the IL-25R might contain phosphorylated tyrosines even in the basal state. In line with this hypothesis, the Y355F IL-25R mutant showed reduced basal tyrosine phosphorylation when transduced into Il-17rb –/– cells. This suggests that constitutive phosphorylation at Tyr-355 may represent a critical impediment for IL-25R activity.
The identity of the tyrosine kinase is being investigated. In silico analyses suggest that
127 janus kinases (JAK2 and JAK3) as well as growth factor receptor kinases such as vascular endothelial growth factor (VEGFR) and platelet derived growth factor
(PDGFR) have predicted sites on the IL-25R. Our preliminary results show constitutive interaction of JAK2 with IL-25R (unpublished observations). Thus, it is possible that JAK2 phosphorylates Tyr-355 in untreated cells, which attracts
DAZAP2 to the IL-25R. What is noteworthy is that P-Tyr of IL-25R increased with ligand stimulation. In a separate study, we found that STAT5 is recruited to IL-25R through the recognition of another tyrosine in a TRAF4-dependent but ACT1- independent manner. Thus, it is conceivable that the removal of DAZAP2 by the
TRAF4-SMURF2 axis also facilitates the phosphorylation of other tyrosines by
JAK2, thereby recruiting the SH2-domain containing STAT5. Future studies are required to define the role of each potential tyrosine on the IL-25R, as each may serve a function in cellular signaling or impinge on receptor conformation.
The complexity of the regulation imposed on the IL-25R underscores the impact of IL-25 in mediating pathological inflammation. Unlike other type-2 cytokines such as IL-13, IL-25 has a profound capacity to initiate the type-2 response.
This activity is due in part to the abundance of IL-25R across multiple cell-types and tissues. Further, in settings of allergic inflammation, IL-25 activity is compounded by the fact that it signals to naïve and activated T-cells to promote Th2 cytokine production (Tamachi et al., 2006;Angkasekwinai et al., 2007;Angkasekwinai et al.,
2010;Swaidani et al., 2011). Thus, given the importance of IL-25, it is not surprising that its receptor is strictly regulated. It would be interesting to determine how
128 DAZAP2 is transcriptionally regulated. Indeed, promoter methylation resulting in
DAZAP2 down-regulation has been described in multiple myeloma; perhaps an aberrant down-regulation of DAZAP2 is present in atopic asthmatics (Shi et al.,
2007;Luo et al., 2012). Furthermore, identifying potential tyrosine kinases or the status of the phosphorylated IL-25R may serve as important therapeutic targets or potential biomarkers, respectively. In summary, this study defined an IL-25R-specific regulatory axis that controls the cellular response to IL-25, which has broad implications in airway inflammation and allergic Type-2 responses.
129
Chapter 4:
Conclusions and future study
130 I. Introduction
Using biochemical and genetic approaches we found that TRAF4 exhibits non- redundant functions in IL-17 and IL-25 signaling pathways. Our studies demonstrated that the mode of TRAF4 engagement is different between IL-17 and
IL-25 signaling pathways. While TRAF4 interacts with Act1 in the IL-17 pathway, IL-
25 stimulation leads to TRAF4 binding to the IL-25R and recruitment of Smurf2.
Importantly, we further establish the central role of TRAF4 in cytokine-elicited as well as allergic airway inflammation. Together these studies will provide further insight towards pathway-specific drug design.
131
II. TRAF4 in the IL-17 signaling pathway
We found that TRAF4-deficiency led to markedly enhanced IL-17 responses.
Direct injection of IL-17 into the peritoneum of Traf4 –/– mice leads to a systemic
increase of chemokine production. Moreover, when we administer intratracheal IL-17 to
Traf4 –/– mice, there is enhanced BAL cellularity and cytokine/chemokine expression in
the lungs. Further, in an IL-17-driven autoimmune model of multiple sclerosis (EAE),
Traf4 –/– mice are more susceptible to disease pathogenesis. Astrocytes are a key IL-17-
responsive cell-type in the CNS. Indeed, astrocytes obtained from Traf4 –/– mice are
hyper-responsive to IL-17. Together the in vivo data suggest that IL-17 responses are
enhanced in the absence of TRAF4.
Next, we examined IL-17-activated signaling pathways in TRAF4-deficient cells.
In the Traf4 –/– kidney epithelial cells, we observed enhanced IL-17-induced phosphorylation and degradation of IκBα. Through co-immunoprecipitation analysis we found that Act1 forms an IL-17-induced complex with TRAF4. This complex was distinct from Act1/TRAF3 or Act1/TRAF6 complexes. In Traf4 –/– cells we noted that
TRAF6 was highly modified and its Act1-interaction was enhanced. Accounting for the reduced NFκB activation, we demonstrate that TRAF4 binds to Act1 via the TRAF- binding-sites, which are also utilized by TRAF6. Thus TRAF4 has the capacity to compete with TRAF6 for Act1 engagement. Taken together, TRAF4 is an Act1- interacting protein that restricts TRAF6-dependent NFκB activation and IL-17-mediated disease pathogenesis.
132
III. TRAF4/SMURF2 in the IL-25 pathway
We identified a unique regulatory axis in the IL-25R pathway. While screening for
TRAF involvement in the IL-25 pathway, we found that TRAF4 was required for IL-25 responses in primary kidney epithelial cells. Furthermore, upon intra-tracheal administration of IL-25 to Traf4 –/– mice we observed significant decreased responses.
There were less eosinophils recruited to the lungs and less IL-25-induced type-2 cytokines such as Il-4, Il-5, Il-13, eotaxin and Il-25. Next, we corroborated these findings in known IL-25 target cells, T-cell and airway epithelial cells. While IL-25 treated T-cells from WT mice produced robust IL-5 and IL-13, this was significantly reduced in the T- cells derived from Traf4 –/– mice. Importantly, the impaired cytokine production was only seen in the IL-25-conditions and not in the IL-4-polarized T-cells. This suggests that there is no global defect in type-2 cytokine production in the absence of TRAF4.
Moreover, T-cells stimulated with IL-25 for short timepoints revealed a necessity for
TRAF4 to activate ERK1/2, P38 as well as STAT5.
Next, we identified a mechanism by which TRAF4 could regulate the IL-25R but not the IL-17A receptor. Two IL-25R-specific binding proteins were identified previously, which included SMURF2 and DAZAP2. Indeed, we confirmed that TRAF4 and
SMURF2 are interacting partners. Furthermore, in Smurf2 –/– cells we observed a reduction in ACT1/IL-25R engagement. In line with this Smurf2 –/– mice have impaired
Type-2 cytokine production and airway eosinophilia in response to IL-25 injection. These results suggested that SMURF2, like TRAF4, is a positive mediator of ACT1 recruitment
133 and of the IL-25 response. SMURF2 is an E3-ubiquitin ligase and DAZAP2 was identified as one of its targets. Given that SMURF2/TRAF4 are positive regulators of the IL-25 response, we hypothesized that DAZAP2 would impose an inhibitory action on the IL-25R. Knockdown of Dazap2 in T-cells or in MEF cell lines resulted in enhance
IL-25-induced type-2 cytokine expression and ACT1/IL-25R interaction respectively.
We observed that IL-25 stimulation led to a concomitant reduction in DAZAP2 protein levels in T-cells. Moreover this reduction was TRAF4/SMURF2 dependent. By co- immunoprecipitation studies, DAZAP2 was K48-linked ubiquitinated following IL-25 stimulation. Thus these data implicate IL-25-induced degradation of the negative regulator, DAZAP2, may be an essential step in order for IL-25 responses to commence.
Finally, we find that DAZAP2 interacts with the IL-25R via its SH2 domains.
Moreover, the IL-25R was precipitated using P-Tyr antibodies, suggesting that the IL-
25R harbors phosphorylated tyrosine residues. Moreover, within the DAZAP2 binding region of the IL-25R is one potential tyrosine residue at site 355. Site-specific mutation of this residue to phenylalanine abrogates the DAZAP2 interaction. Furthermore, restoration of IL-25R-deficient cells with this y355f mutant leads to enhanced IL-25 response. Together, these data indicate that phosphorylated tyrosine within the IL-25R is critical for DAZAP2 binding and imposition of its inhibitory activity.
134 IV. Future study
A. TRAF4 as a therapeutic target in inflammatory diseases
A growing body of evidence suggests that TRAF proteins delineate the signaling outcome following IL-17-receptor activation. For example, in the IL-17 pathway
TRAF6/Act1 interaction is required for NFκB activation, TRAF2/TRAF5/Act1 interaction controls the stability of chemokine mRNA (Hartupee et al., 2009; Bulek et al.,
2011; Sun et al., 2011). Based on our studies, TRAF4 would be an intriguing target for therapeutic intervention in airway diseases. Targeting TRAF4 would leave the IL-17- response intact whilst blocking IL-25-driven eosinophilia and mucus production. In the airways, IL-17 is considered to be a critical cytokine for innate immune response to extracellular bacteria and fungus. In line with this, blockade of IL-17 in humans has actually been detrimental to the hosts’ response to these pathogens. One recent example was a clinical study in which IL-17 was targeted with monoclonal antibody treatment in patients with Crohn’s disease, an inflammatory gastrointestinal disorder (Hueber et al.,
2012). Blockade of IL-17 led to adverse consequences including susceptibility to fungal infections. Ultimately the trial was terminated. We may predict a similar outcome when treating asthmatics treated with anti-IL-17A. Since IL-25 is considered to be a major driver/initiator of Type-2 inflammation it remains an attractive therapeutic target in asthmatics. Our studies indicate that TRAF4 is critical for IL-25-induced production of
IL-5, IL-13 and mucus in an allergic airway inflammation model. Moreover, studies have shown that therapeutic intervention by blockade of IL-25 in mice, alleviates type-2 inflammation and airway hyper-responsiveness (Ballantyne et al., 2007). In theory, small
135 molecule inhibitors of the TRAF4/IL-25R interaction could effectively block IL-25- driven responses without disrupting the beneficial activities of IL-17A in normal host defense.
B. IL-25R-dependent STAT activation
One major finding is that the IL-25R may in fact harbor phosphorylated tyrosines within its intracellular region. Moreover, mutation of a single tyrosine to a phenylalanine had a drastic effect on the IL-25 response. Thus, further evaluation of the other six individual tyrosines and how they impact IL-25 responses is certainly warranted. Our observations provide the first indication that an IL-17R family-member may be a direct target of phosphorylation and that this event impinges upon its activity. Numerous cytokine receptors such as those activated by IL-2, IL-4, IL-13 and Interferon-Gamma, to name a few, activate downstream signaling molecules known as Janus Kinases (JAKs) and the transcription factor family– STATs (Signal Transducer and Activator of
Transcription). Upon ligand-receptor binding JAK-kinase activity is activated, leading to phosphorylation of tyrosine residues within the receptor. The phosphorylated receptor is then recognized by STAT proteins, which are subsequently phosphorylated by JAK. The
P-STAT undergoes dimerization and then translocates to the nucleus for target gene transcription. Current work from our lab has found that IL-25 can induce the phosphorylation of STAT5 in T-cells and its association with the IL-25R (Fig. 4-1a-b).
Importantly this event is dependent upon TRAF4 (Fig. 4-1a-b).
136
Figure 4-1. TRAF4 is required for P-STAT5 activation. (a) Primary murine T-cells isolated from WT and Traf4 –/– mice were polarized in Th2 conditions for 4 days after which they were stimulated for the indicated times with IL-25 (100 ng/ml). Lysates were collected and subjected to immunoblot analysis. Shown is a representative experiment (same as shown in Figure 3-5). (b) Primary murine kidney epithelial cell transduced with Ad-V5-IL-25R were treated with IL-25, lysate was prepared and co-immunoprecipitated with Anti-V5 followed by immunoblot analysis. A representative result from three independent experiments is shown.
Since IL-25 is known to drive the expression of type-2 cytokines (Il-4, Il-5 and
Il-13), one hypothesis is that the expression of these cytokines depends on IL-25- induced activation of STAT5. Several reports have implicated STAT5 as critical transcription factor for type-2 cytokines, including IL-4 (Zhu et al., 2002). These studies have shown that STAT5 interacts with the promoter region of these
137 cytokines, thereby leading to an open conformation of the DNA that promotes further recruitment of other transcription factors critical for type-2 signature gene expression (Zhu et al., 2002). Thus future studies will need to be conducted in order to determine how IL-25-induced STAT5 activation and Act1 may cooperate to control type-2 responses. Furthermore, a detailed mapping of the responsible P-Tyr on the IL-25R and the kinase will be insightful and could be exploited for therapeutic intervention.
C. The IL-25R as a therapeutic target or potential biomarker in airway disorders
Several reports now show that the IL-25R is highly expressed in lung tissue and eosinophils derived from asthmatics (Wang et al., 2007;Tang et al., 2014). Due to
IL-25’s capacity to initiate and propagate type-2 cytokine production, blockade of IL-
25 and its receptor are attractive targets for therapeutic intervention. In the OVA- asthma mouse model, IL-25-specific monoclonal antibody treatment, leads to a reduction in airway hyper-reactivity (AHR) as well as IL-13 production (Ballantyne et al., 2007; Rickel et al., 2008). These observations suggest that blockade of the IL-25R may have a similar protective effect during an allergic airway response.
The observations made in our study revealed that the IL-25R might be phosphorylated, which may be indicative of IL-25R activity. Although we only evaluated an inhibitory tyrosine residue (355) in our study, it is important to note that other tyrosines may actually be required for IL-25 responses. Thus, developing
138 antibodies that specifically recognize phosphorylated forms of the IL-25R could be used as diagnostic markers. Although, not addressed in our study, potential kinases that phosphorylate the IL-25R should also be evaluated. Screening for potential kinases using available databases revealed that JAK family members such as JAK2 as well as growth factor receptor tyrosine kinases and EphB-family of kinases have predicted sites on the IL-25R. Once the function of these kinases is determined, they would be invaluable exploits for therapy.
Final remarks
The responses initiated by cytokines during inflammatory reactions must be tightly regulated in order to preserve the host tissue from adverse damage. It is becoming increasingly clear that autoimmune diseases such as multiple sclerosis and chronic diseases like asthma are perpetuated by an uncontrolled bout of inflammation. The IL-17-cytokine family is abundantly expressed across multiple tissues and exhibit extraordinary pleiotropic activities. Indeed, compared to IL-17A,
IL-25 (IL-17E) is by far the most structurally and functionally divergent. Moreover, these cytokines have directly been shown to mediate the hallmark disease pathology associated with multiple sclerosis and airway inflammation.
By identifying the unique requirements for signaling molecules downstream of the IL-17 and IL-25 receptor we have advanced our understanding of how and why these cytokines work differently. Although we’ve known that ACT1 is required for an effective IL-17 or an IL-25 response, the distinguishing features leading to their non- redundant biological activities were entirely unknown. Building upon our knowledge
139 that TRAF molecules mediate specificity in the IL-17 response we uncovered a unique and non-redundant function of TRAF4 in the IL-17 vs. IL-25 pathways. In the IL-17 pathway TRAF4 interacted with ACT1 thereby inhibiting ACT1/TRAF6 ineraction and subsequent NFkB activation. Unlike the IL-17 pathway, TRAF4 could interact directly with the IL-25R in an ACT1-independent manner. TRAF4- deficiency, in IL-25-target cells as well as in in vivo models, abolished the IL-25 response. At the core of TRAF4-IL-25 activity, was a unique inhibitory molecule,
DAZAP2. We defined the requirement for TRAF4-dependent SMURF2-mediated
DAZAP2 degradation as an IL-25-elicited permissive event required for an effective
IL-25 response. We further identified the IL-25R as containing potential phosphorylated tyrosines. Together, not only do these findings warrant further investigation for therapeutic targets, but they also offer new paradigms to be explored and built upon for IL-17 cytokine biology.
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