Shadi Swaidani

Nitrotyrosine Proteome Survey in Asthma Identifies Oxidative Mechanism of Catalase Inactivation

Reactive oxygen species and reactive nitrogen species produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Detection of 3-nitrotyrosine in the asthmatic lung confirms the presence of increased reactive oxygen and nitrogen species, but the lack of identification of modified proteins has hindered an understanding of the potential mechanistic contributions of nitration/oxidation to airway inflammation. In this study, we applied a proteomic approach, using nitrotyrosine as a marker, to evaluate the oxidation of proteins in the allergen-induced murine model of asthma. Over 30 different proteins were targets of nitration following allergen challenge, including the antioxidant enzyme catalase. Oxidative modification and loss of catalase enzyme function were seen in this model. Subsequent investigation of human bronchoalveolar lavage fluid revealed that catalase activity was reduced in asthma by up to 50% relative to healthy controls. Analysis of catalase isolated from asthmatic airway epithelial cells revealed increased amounts of several protein oxidation markers, including chloro- and nitrotyrosine, linking oxidative modification to the reduced activity in vivo. Parallel in vitro studies using reactive chlorinating species revealed that catalase inactivation is accompanied by the oxidation of a specific cysteine (Cys377). Taken together, these studies provide evidence of multiple ongoing and profound oxidative reactions in asthmatic airways, with one early downstream consequence being catalase inactivation. Loss of catalase activity likely amplifies oxidative stress, contributing to the chronic inflammatory state of the asthmatic airway.

Act1, a U-box E3 ubiquitin ligase for IL-17 signaling.

The adaptor protein Act1 functions as a ubiquitin ligase to mediate interleukin-17 receptor–dependent activation of nuclear factor-κB. Acting Out a New Role Interleukin-17A (IL-17A) is the founding member of a family of proinflammatory cytokines implicated in the development of autoimmunity and the fight against microbial infection. IL-17 signaling, which is mediated by a number of IL-17 receptor (IL-17R) complexes (see the Perspective by Levin), depends on TRAF6, a scaffold protein and E3 ubiquitin ligase, and activates the transcription factor NF-κB. TRAF6 is recruited to IL-17Rs through another adaptor protein, Act1. Liu et al. now show that Act1 is also a member of the U-box family of E3 enzymes and that its ubiquitin ligase activity is required for IL-17R signaling. In response to IL-17, TRAF6 was recruited to Act1 and underwent Lys63-mediated ubiquitination, a process that was required for the activation of NF-κB. A mutant form of TRAF6 lacking the residue targeted by ubiquitin failed to support IL-17–mediated activation of NF-κB in TRAF6-deficient cells. Together, these data identify TRAF6 as a substrate of Act1 and suggest that sequential ubiquitination events regulate IL-17–induced inflammation. Interleukin-17 (IL-17), a proinflammatory cytokine mainly produced by cells of the T helper 17 (TH17) lineage, is required for host defense against bacterial and fungal infections and plays a critical role in the pathogenesis of inflammatory and autoimmune diseases. Act1 is an essential adaptor molecule in IL-17–mediated signaling and is recruited to the IL-17 receptor (IL-17R) upon IL-17 stimulation through an interaction between its SEFIR domain and that of the IL-17R. Here, we report that Act1 is a U-box E3 ubiquitin ligase and that its activity is essential for IL-17–mediated signaling pathways. Through the use of the Ubc13-Uev1A E2 complex, Act1 mediated the lysine-63–linked ubiquitination of tumor necrosis factor receptor–associated factor 6 (TRAF6), a component of IL-17–mediated signaling. Deletion and point mutations of the Act1 U-box abolished Act1-mediated ubiquitination of TRAF6 and impaired the ability of Act1 to restore IL-17–dependent signaling and expression of target genes in Act1−/− mouse embryonic fibroblasts. We also showed that the lysine-124 residue of TRAF6 was critical for efficient Act1-mediated ubiquitination of TRAF6 and for the ability of TRAF6 to mediate IL-17–induced activation of nuclear factor κB. Thus, we propose that Act1 mediates IL-17–induced signaling pathways through its E3 ubiquitin ligase activity and that TRAF6 is a critical substrate of Act1, which indicates the importance of protein ubiquitination in the IL-17–dependent inflammatory response.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1–TRAF6–transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Epithelium: the interplay between innate and Th2 immunity

Mucosal epithelium functions not only as a physical barrier, but also as a regulator of innate and adaptive immune responses against foreign substances and microorganisms. In particular, epithelial cells have been directly implicated in Th2 responses, serving as a critical interface between innate immune responses and Th2 immunity. Emerging studies have revealed the cellular and molecular mechanisms by which the epithelium modulates Th2 responses through the production of a group of epithelial‐derived Th2‐driving cytokines, including interleukin (IL)‐25, IL‐33, and thymic stromal lymphopoietin. These epithelial‐derived Th2‐driving cytokines execute a regulatory function of the epithelium on mucosal immunity by promoting Th2 responses and maintaining the balance of host immune homeostasis and defense against various pathogens. Dysregulation of these Th2‐driving cytokines can lead to detrimental Th2‐dependent inflammatory responses, often manifested in various forms of allergic and inflammatory diseases.

Th1- and Th2-Dependent Endothelial Progenitor Cell Recruitment and Angiogenic Switch in Asthma

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.

Epithelial Cell-Specific Act1 Adaptor Mediates Interleukin-25-Dependent Helminth Expulsion through Expansion of Lin−c-Kit+ Innate Cell Population

Interleukin-25 (IL-25 or IL-17E), a member of the structurally related IL-17 family, functions as an important mediator of T helper 2 cell-type (type 2) responses. We examined the cell type-specific role of IL-25-induced Act1-mediated signaling in protective immunity against helminth infection. Targeted Act1 deficiency in epithelial cells resulted in a marked delay in worm expulsion and abolished the expansion of the Lin(-)c-Kit(+) innate cell population in the mesenteric lymph node, lung, and liver. Th2 cell-inducing cytokine (IL-25 and IL-33) expression were reduced in the intestinal epithelial cells from the infected and IL-25-injected epithelial-specific Act1-deficient mice. Adoptive transfer of Lin(-)c-Kit(+) cells or combined injection of IL-25 and IL-33 restored the type 2 responses in these mice. Taken together, these results suggest that epithelial-specific Act1 mediates the expansion of the Lin(-)c-Kit(+) innate cell population through the positive-feedback loop of IL-25, initiating the type 2 immunity against helminth infection.

Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma.

Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24 h of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2 h of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

Background: TSG-6 transfers heavy chains (HCs) from the inter-α-inhibitor to hyaluronan (HA), increasing its avidity for leukocytes. Results: Recombinant TSG-6 increased leukocyte adhesion to HA and its accumulation in airway cells. Conclusion: In addition to its ability to transfer HCs to HA, TSG-6 amplifies HA synthesis. Significance: TSG-6 is a potent regulator of HA synthesis and is likely to be involved in a variety of inflammatory diseases. We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6−/−, HAS1/3−/−, and CD44−/− mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

TSG-6 protein is crucial for the development of pulmonary hyaluronan deposition, eosinophilia, and airway hyperresponsiveness in a murine model of asthma.

Background: TSG-6 is important in the organization of hyaluronan (HA). Results: Lack of TSG-6 results in diminished HA accumulation, inflammation, and airway hyperresponsiveness. Conclusion: TSG-6 is essential for the pathological manifestations in a murine model of asthma. Significance: TSG-6 is likely to contribute to the pathogenesis of asthma. Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.

A CC′ Loop Decoy Peptide Blocks the Interaction Between Act1 and IL-17RA to Attenuate IL-17– and IL-25–Induced Inflammation

A cell-permeable peptide derived from an adaptor protein blocks cytokine receptor signaling and reduces inflammation. Quelling Inflammation with a Peptide Members of the interleukin-17 (IL-17) family of cytokines, including IL-17A and IL-25, are implicated in the pathogenesis of various inflammatory conditions, including allergen-induced pulmonary inflammation. Indeed, the concentrations of IL-17A and IL-25 are higher in the lungs of asthmatic patients than in those of unaffected individuals. IL-17A and IL-25 share a common receptor subunit, IL-17RA, to which is recruited the adaptor protein Act1 through interactions between its SEFIR domain and the SEFIR domain of the receptor. Liu et al. identified the CC′ loop region within the SEFIR domain of Act1 as critical for receptor-adaptor protein interactions. A cell-permeable peptide based on this region inhibited IL-17– and IL-25–dependent signaling in vitro and reduced disease in a mouse model of pulmonary inflammation, suggesting that such a strategy may provide therapies to treat IL-17–mediated inflammatory diseases in humans. Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll–IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB′ loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB′ loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1–IL-17RA interactions; rather, deletion of the CC′ loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC′ loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC′ loop sequence inhibited IL-17– or IL-25–mediated signaling in vitro, as well as IL-17– and IL-25–induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC′ loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.