Interleukin-13 Propagates Prothrombin Kringle-2-Induced Neurotoxicity in Hippocampi In Vivo via Oxidative Stress.

The present study investigated expression of endogenous interleukin-13 (IL-13) and its possible function in the hippocampus of prothrombin kringle-2 (pKr-2)-lesioned rats. Here we report that intrahippocampal injection of pKr-2 revealed a significant loss of NeuN-immunopositive (NeuN+) and Nissl+ cells in the hippocampus at 7 days after pKr-2. In parallel, pKr-2 increased IL-13 levels, which reached a peak at 3 days post pKr-2 and sustained up to 7 days post pKr-2. IL-13 immunoreactivity was seen exclusively in activated microglia/macrophages and neutrophils, but not in neurons or astrocytes. In experiments designed to explore the involvement of IL-13 in neurodegeneration, IL-13 neutralizing antibody (IL-13Nab) significantly increased survival of NeuN+ and Nissl+ cells. Accompanying neuroprotection, immunohistochemical analysis indicated that IL-13Nab inhibited pKr-2-induced expression of inducible nitric oxide synthase and myeloperoxidase within activated microglia/macrophages and neutrophils, possibly resulting in attenuation of reactive oxygen species (ROS) generation and oxidative damage of DNA and protein. The current findings suggest that the endogenous IL-13 expressed in pKr-2 activated microglia/macrophages and neutrophils might be harmful to hippocampal neurons via oxidative stress.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

Th2 cytokines orchestrate the secretion of MUC5AC and MUC5B in IL-5-positive chronic rhinosinusitis with nasal polyps.

BACKGROUND: Mucin over-secretion is a significant characteristic of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to investigate the relationship between Th2 cytokines and MUC5AC or MUC5B, and the mechanism of mucin over-secretion in the type-2 inflammatory endotype of CRSwNP. METHODS: Main Th-cell cytokines, associated mediators, and mucins were determined in the homogenates of nasal polyp samples from 21 CRSwNP patients and inferior turbinate samples from 8 controls, by ELISA or UniCAP system. Secretion of MUC5AC and MUC5B was measured in the supernatants of IL-5, IL-4, or IL-13 primed nasal polyp fragments. Co-localization of MUC5AC, MUC5B, and IL-4 receptor α (IL-4Rα) in CRSwNP and controls was evaluated by immunohistochemistry. Gene expression of IL-4Rα in the samples was measured by real-time reverse transcription-polymerase chain reaction. RESULTS: Baseline protein levels of the Th2-cytokines IL-4, IL-5, and IL-13, and mucins MUC5AC and MUC5B were significantly higher in the IL-5(+) CRSwNP group, compared to control and IL-5(-) CRSwNP groups. MUC5AC and MUC5B secretions were significantly increased in IL-4- or IL-13-primed, but not IL-5-primed fragments of nasal polyps. Immuno-stained serial sections demonstrated that IL-4Rα was widely expressed in the epithelium and submucosal glands in control and nasal polyp tissues. Gene expression of IL-4Rα was elevated in nasal polyp tissues, specifically in the IL-5(+) CRSwNP group. CONCLUSIONS: In type-2 inflammatory nasal polyps, characterized by the tissue expression of IL-5, MUC5AC and MUC5B are overexpressed. Both IL-4 and IL-13 may upregulate mucin expression via IL-4Rα, which is also overexpressed in IL-5(+) CRSwNP.

Regulation of monoamine oxidase A (MAO-A) expression, activity, and function in IL-13-stimulated monocytes and A549 lung carcinoma cells.

Monoamine oxidase A (MAO-A) is a mitochondrial flavoenzyme implicated in the pathogenesis of atherosclerosis and inflammation and also in many neurological disorders. MAO-A also has been reported as a potential therapeutic target in prostate cancer. However, the regulatory mechanisms controlling cytokine-induced MAO-A expression in immune or cancer cells remain to be identified. Here, we show that MAO-A expression is co-induced with 15-lipoxygenase (15-LO) in interleukin 13 (IL-13)-activated primary human monocytes and A549 non-small cell lung carcinoma cells. We present evidence that MAO-A gene expression and activity are regulated by signal transducer and activator of transcription 1, 3, and 6 (STAT1, STAT3, and STAT6), early growth response 1 (EGR1), and cAMP-responsive element-binding protein (CREB), the same transcription factors that control IL-13-dependent 15-LO expression. We further established that in both primary monocytes and in A549 cells, IL-13-stimulated MAO-A expression, activity, and function are directly governed by 15-LO. In contrast, IL-13-driven expression and activity of MAO-A was 15-LO-independent in U937 promonocytic cells. Furthermore, we demonstrate that the 15-LO-dependent transcriptional regulation of MAO-A in response to IL-13 stimulation in monocytes and in A549 cells is mediated by peroxisome proliferator-activated receptor γ (PPARγ) and that signal transducer and activator of transcription 6 (STAT6) plays a crucial role in facilitating the transcriptional activity of PPARγ. We further report that the IL-13-STAT6-15-LO-PPARγ axis is critical for MAO-A expression, activity, and function, including migration and reactive oxygen species generation. Altogether, these results have major implications for the resolution of inflammation and indicate that MAO-A may promote metastatic potential in lung cancer cells.

Anti-inflammatory (M2) macrophage media reduce transmission of oligomeric amyloid beta in differentiated SH-SY5Y cells.

Neuroinflammation plays an influential role in Alzheimer's disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oAß) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oAß, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oAß and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies.

IL-13 from intraepithelial lymphocytes regulates tissue homeostasis and protects against carcinogenesis in the skin.

The skin is under constant renewal and exposure to environmental challenges. How homeostasis is maintained alongside protective mechanisms against damage is unclear. Among the basal epithelial cells (ECs) is a population of resident intraepithelial lymphocytes (IELs) that provide host-protective immune surveillance. Here we show that IELs cross-communicate with ECs via the production of IL-13. Skin ECs are activated by IEL-derived IL-13, enabling a canonical EC stress response. In the absence of IL-13, or canonical IEL, the skin has decreased ability to repair its barrier and increased susceptibility to cutaneous carcinogenesis. IL-13 controls the rate of EC movement through the epidermis, which might explain the importance of IL-13 for epidermal integrity and its suppressive effect on skin carcinogenesis. These findings show that IL-13 acts as a molecular bridge between IELs and ECs, and reveal a critical host-defensive role for type-2 immunity in regulating EC tissue homeostasis and carcinogenesis.

IL-13 Augments Compressive Stress-Induced Tissue Factor Expression in Human Airway Epithelial Cells.

Tissue factor (TF) is best known as a cellular initiator of coagulation, but it is also a multifunctional protein that has been implicated in multiple pathophysiologic conditions, including asthma. In the lung, airway epithelial cells express TF, but it is unknown how TF expression is regulated by asthma-associated mediators. We investigated the role of IL-13, a type 2 cytokine, alone and in combination with compressive stress, which mimics asthmatic bronchoconstriction, on TF expression and release of TF-positive extracellular vesicles from primary normal human bronchial epithelial cells. Well-differentiated normal human bronchial epithelial cells were treated with IL-13 and compressive stress, alone and in combination. TF mRNA, protein and activity were measured in the cells and conditioned media. TF was also measured in the bronchoalveolar lavage (BAL) fluid of allergen-challenged mice and patients with asthma. IL-13 and compressive stress increased TF expression, but only compressive stress induced TF-positive extracellular vesicle release. Pretreatment with IL-13 augmented compressive stress-induced TF expression and release. TF protein and activity in BAL fluid were increased in allergen-sensitized and -challenged mice. TF was elevated in the BAL fluid of patients with mild asthma after an allergen challenge. Our in vitro and in vivo data indicate close cooperation between mechanical and inflammatory stimuli on TF expression and release of TF-positive extracellular vesicles in the lungs, which may contribute to pathophysiology of asthma.

Modulation of pulmonary fibrosis by IL-13Rα2.

Pulmonary fibrosis is a progressive and fatal disease that involves the remodeling of the distal airspace and the lung parenchyma, which results in compromised gas exchange. The median survival time once diagnosed is less than three years. Interleukin (IL)-13 has been shown to play a role in a number of inflammatory and fibrotic diseases. IL-13 modulates its effector functions via a complex receptor system that includes the IL-4 receptor (R) α, IL-13Rα1, and the IL-13Rα2. IL-13Rα1 binds IL-13 with low affinity, yet, when it forms a complex with IL-4α, it binds with much higher affinity, inducing the effector functions of IL-13. IL-13Rα2 binds IL-13 with high affinity but has a short cytoplasmic tail and has been shown to act as a nonsignaling decoy receptor. Transfection of fibroblasts and epithelial cells with IL-13Rα2 inhibited the IL-13 induction of soluble collagen, TGF-ß, and CCL17. Adenoviral overexpression of IL-13Rα2 in the lung reduced bleomycin-induced fibrosis. Our work shows that overexpression of IL-13Rα2 inhibits the IL-13 induction of fibrotic markers in vitro and inhibits bleomycin-induced pulmonary fibrosis. In summary our study highlights the antifibrotic nature of IL-13Ra2.

CXCR3 Requirement for the Interleukin-13-Mediated Up-Regulation of Interleukin-13Rα2 in Pulmonary Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by fibrosis and abnormal vascularity. IL-13, a profibrotic cytokine that plays a role in IPF, functions through the Jak/STAT pathway after binding to the IL-13 receptor α1 (IL-13Rα1)/IL-4Rα complex. IL-13 also binds to IL-13Rα2, which has been thought to function as a nonsignaling decoy receptor, although possible signaling roles of this receptor have been proposed. CXCR3 and its IFN-inducible ligands-CXCL9, CXCL10, and CXCL11-have been implicated in vascular remodeling and fibroblast motility during the development of IPF. In this study, CXCR3 expression was demonstrated in cultured pulmonary fibroblasts from wild-type BALB/c mice and was found to be necessary for the IL-13-mediated gene and protein up-regulation of IL-13Rα2. In fibroblasts from CXCR3-deficient mice, STAT6 activation was prolonged. This study is the first to demonstrate the expression of CXCR3 in fibroblasts and its association with the expression of IL-13Rα2. Taken together, the results from this study point strongly to a requirement for CXCR3 for IL-13-mediated IL-13Rα2 gene expression. Understanding the function of CXCR3 in IL-13-mediated lung injury may lead to novel approaches to combat the development of pulmonary fibrosis, whether by limiting the effects of IL-13 or by manipulation of angiostatic pathways. The elucidation of the complex relationship between these antifibrotic receptors and manipulation of the CXCR3-mediated regulation of IL-13Rα2 may represent a novel therapeutic modality in cases of acute lung injury or chronic inflammation that may progress to fibrosis.

Adipocyte-specific IKKß signaling suppresses adipose tissue inflammation through an IL-13-dependent paracrine feedback pathway.

Adipose tissue inflammation is one pathway shown to mediate insulin resistance in obese humans and rodents. Obesity induces dynamic cellular changes in adipose tissue to increase proinflammatory cytokines and diminish anti-inflammatory cytokines. However, we have found that anti-inflammatory interleukin-13 (IL-13) is unexpectedly induced in adipose tissue of obese humans and high-fat diet (HFD)-fed mice, and the source of IL-13 is primarily the adipocyte. Moreover, HFD-induced proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1ß mediate IL-13 production in adipocytes in an IKKß-dependent manner. In contrast, adipocyte-specific IKKß-deficient mice show diminished IL-13 expression and enhanced inflammation after HFD feeding, resulting in a worsening of the insulin-resistant state. Together these data demonstrate that although IKKß activates the expression of proinflammatory mediators, in adipocytes, IKKß signaling also induces the expression of the anti-inflammatory cytokine IL-13, which plays a unique protective role by limiting adipose tissue inflammation and insulin resistance.

Biliary repair and carcinogenesis are mediated by IL-33-dependent cholangiocyte proliferation.

Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.

Protection against type 1 diabetes upon Coxsackievirus B4 infection and iNKT-cell stimulation: role of suppressive macrophages.

Invariant natural killer T (iNKT) cells belong to the innate immune system and exercise a dual role as potent regulators of autoimmunity and participate in responses against different pathogens. They have been shown to prevent type 1 diabetes development and to promote antiviral responses. Many studies in the implication of environmental factors on the etiology of type 1 diabetes have suggested a link between enteroviral infections and the development of this disease. This study of the pancreatropic enterovirus Coxsackievirus B4 (CVB4) shows that although infection accelerated type 1 diabetes development in a subset of proinsulin 2-deficient NOD mice, the activation of iNKT cells by a specific agonist, α-galactosylceramide, at the time of infection inhibited the disease. Diabetes development was associated with the infiltration of pancreatic islets by inflammatory macrophages, producing high levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and activation of anti-islet T cells. On the contrary, macrophages infiltrating the islets after CVB4 infection and iNKT-cell stimulation expressed a number of suppressive enzymes, among which indoleamine 2,3-dioxygenase was sufficient to inhibit anti-islet T-cell response and to prevent diabetes. This study highlights the critical interaction between virus and the immune system in the acceleration or prevention of type 1 diabetes.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma.

PURPOSE: In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN: We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS: Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS: A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

Anti-IL-13 in inflammatory bowel disease: from the bench to the bedside.

Much work has been done to understand the molecular mechanisms underlying the inflammatory bowel diseases (IBD). IL-13 has emerged as an important cytokine effective in ulcerative colitis (UC) and fistulizing Crohn's disease (CD). IL-13 is a T helper 2-type cytokine with pleiotropic effects, involved in parasite expulsion, asthma pathophysiology, natural history of cancer and other human pathologies. Great interest has therefore been developed in inhibiting its function as a therapeutic intervention in these diseases. The multifunctional properties of IL-13, with particular emphasis on its role in both CD and UC, as well as current developing pharmacologic agents inhibiting the IL-13 signaling pathway have been reviewed. Anti-IL-13 agents seem to be promising therapeutic strategies for the future management of IBD and other human diseases.

Dynamic mast cell-stromal cell interactions promote growth of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment, which includes cancer-associated fibroblasts [also known as pancreatic stellate cells (PSC)] and immune cells that provide a fibrotic niche that impedes successful cancer therapy. We have found that mast cells are essential for PDAC tumorigenesis. Whether mast cells contribute to the growth of PDAC and/or PSCs is unknown. Here, we tested the hypothesis that mast cells contribute to the growth of PSCs and tumor cells, thus contributing to PDAC development. Tumor cells promoted mast cell migration. Both tumor cells and PSCs stimulated mast cell activation. Conversely, mast cell-derived interleukin (IL)-13 and tryptase stimulated PSC proliferation. Treating tumor-bearing mice with agents that block mast cell migration and function depressed PDAC growth. Our findings suggest that mast cells exacerbate the cellular and extracellular dynamics of the tumor microenvironment found in PDAC. Therefore, targeting mast cells may inhibit stromal formation and improve therapy.

Signal transducer and activator of transcription 6 directly regulates human ORMDL3 expression.

Orosomucoid-like 3 (ORMDL3) has been associated with asthma and a series of autoimmune disorders, and is involved in endoplasmic reticulum-mediated inflammatory responses. However, its clinical significance and the molecular mechanism underlying its expression are still largely unclear. To elucidate the mechanisms of human ORMDL3 transcriptional regulation, we cloned a 1.5 kb genomic DNA fragment containing the putative promoter region and evaluated its transcriptional activity in a luciferase reporter system by deletion analysis. We identified a 68 bp region that functions as a minimal promoter. Bioinformatics analysis predicted that the -64 to -56 bp region contained a signal transducer and activator of transcription 6 (STAT6) binding site. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that STAT6 bound to its binding site within the ORMDL3 promoter. STAT6 over-expression or knockdown trans-activated or trans-inhibited, respectively, the ORMDL3 promoter containing the STAT6-binding motif. Treatment with interleukins 4 or 13 increased ORMDL3 promoter activity as well as endogenous ORMDL3 expression. Immunoprecipitation and ChIP/Re-ChIP assays revealed that STAT6 and p300 exist in the same protein complex that binds to the ORMDL3 promoter. Our study confirmed that STAT6 plays important roles in regulating the expression of human ORMDL3 by directly binding to the promoter region, which may shed light on a possible role in various human diseases.