Therapeutic Evaluation of Antibody-Based Targeted Delivery of Interleukin 9 in Experimental Pulmonary Hypertension.

BACKGROUND AND AIMS: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. METHODS: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. RESULTS: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. CONCLUSIONS: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling.

Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells.

Th9 cells orchestrate allergic lung inflammation by promoting recruitment and activation of eosinophils and mast cells, and by stimulating epithelial mucus production, which is known to be mainly dependent on IL-9. These cells share developmental pathways with induced regulatory T cells that may determine the generation of one over the other subset. In fact, the FOXP3 transcription factor has been shown to bind il9 locus and repress IL-9 production. The microbiota-derived short-chain fatty acids (SCFAs) butyrate and propionate have been described as FOXP3 inducers and are known to have anti-inflammatory properties. While SCFAs attenuate lung inflammation by inducing regulatory T cells and suppressing Th2 responses, their effects on Th9 cells have not been addressed yet. Therefore, we hypothesized that SCFAs would have a protective role in lung inflammation by negatively modulating differentiation and function of Th9 cells. Our results demonstrated that butyrate is more effective than propionate in promoting FOXP3 expression and IL-9 repression. In addition, propionate was found to negatively impact in vitro differentiation of IL-13-expressing T cells. Butyrate treatment attenuated lung inflammation and mucus production in OVA-challenged mice, which presented lower frequency of lung-infiltrated Th9 cells and eosinophils. Both Th9 cell adoptive transfer and IL-9 treatment restored lung inflammation in butyrate-treated OVA-challenged mice, indicating that the anti-inflammatory effects of butyrate may rely on suppressing Th9-mediated immune responses.

Neutralization of interleukin-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Astrocytes mediate the destruction of the blood-brain barrier (BBB) during ischemic stroke (IS). IL-9 is a pleiotropic cytokine that we previously found to be highly expressed in peripheral blood mononuclear cells from patients with IS, and the presence of IL-9 receptors on astrocytes has been reported in the literature. Here, we detected the effect of IL-9 on astrocytes using an anti-IL-9-neutralizing antibody to treat rats with experimental stroke. Supernatants from astrocytes treated with or without oxygen-glucose deprivation and/or IL-9 were incubated with bEnd.3 cell monolayers after blocking the IL-9 receptor on the endothelium. Immunofluorescence staining and Western blot analyses were conducted to observe the change in tight junction proteins (TJPs) in bEnd.3 cells as well as the level of VEGF-A and possible signal pathways in astrocytes. We also applied middle cerebral artery occlusion (MCAO) models to determine the effect of anti-IL-9-neutralizing antibodies on IS. As a result, astrocyte-conditioned medium treated with IL-9 aggravated the disruption of the BBB accomplished by the degradation of TJPs in endothelial cells. In addition, IL-9 increased the level of VEGF-A in astrocytes, and blocking the effect of VEGF-A reversed the breakdown of the BBB. In the MCAO model, anti-IL-9-neutralizing antibody reduced the infarct volume and BBB destruction. Mechanistically, the anti-IL-9-neutralizing antibody repaired the damaged TJPs (zonula occludens 1, occludin, and claudin-5) and induced a decrease in VEGF-A expression in ischemic lateral brain tissue. In contrast, a local injection of recombinant murine IL-9 to the brain resulted in a marked up-regulation of VEGF-A in the striatum. In conclusion, anti-IL-9-neutralizing antibody can reduce the severity of IS partially by alleviating the destruction of the BBB via down-regulation of astrocyte-derived VEGF-A. This finding suggests that targeting IL-9 or VEGF-A could provide a new direction for the treatment of IS.-Tan, S., Shan, Y., Lin, Y., Liao, S., Zhang, B., Zeng, Q., Wang, Y., Deng, Z., Chen, C., Hu, X., Peng, L., Qiu, W., Lu, Z. Neutralization of IL-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Interleukin-9 Promotes Pancreatic Cancer Cells Proliferation and Migration via the miR-200a/Beta-Catenin Axis.

Background. Both IL-9 and miR-200a are involved in the pathogenesis of cancers; however, the role of IL-9 in pancreatic cancer and the possible underlying mechanisms remain unknown. The aim of this study was to investigate the effect of IL-9 on pancreatic cancer cells and its interaction with miR-200a. Methods. Pancreatic cancer cells (PANC-1 and AsPC-1) were treated with IL-9 and the expression of miR-200a and ß-catenin in pancreatic cancer cells was measured. ß-Catenin was examined as a target gene of miR-200a in pancreatic cancer cells. The interaction between IL-9 and miR-200a in pancreatic cancer cells was determined by infecting miR-200a mimics prior to IL-9 treatment and then measuring miR-200a and ß-catenin expression. Results. IL-9 significantly promoted the proliferation, invasion, and migration of pancreatic cancer cells; however, the effect on pancreatic cancer cell apoptosis was insignificant. ß-Catenin was verified as a target gene of miR-200a in pancreatic cancer cells. Overexpression of miR-200a in pancreatic cancer cells significantly attenuated proliferation and metastasis and reduced ß-catenin expression. IL-9 treatment of pancreatic cancer cells decreased miR-200a expression and increased ß-catenin expression. The effect of miR-200a on pancreatic cancer cells decreased following IL-9 treatment. Conclusions. IL-9 promotes proliferation and metastasis in pancreatic cancer cells; this effect may partly involve regulation of the miR-200a/ß-catenin axis.

IL-9 aggravates the development of atherosclerosis in ApoE-/- mice.

AIMS: Recently, interleukin (IL)-9 was found to be involved in the pathogenesis of many inflammatory diseases. Here, we tested whether IL-9 was related to atherosclerosis and investigated the underlying mechanisms. METHODS AND RESULTS: IL-9R was expressed in mouse aortic endothelial cells (MAECs) and aortic tissues, and IL-9 levels were elevated in plasma and aortic arches in Apolipoprotein E-deficient (ApoE-/-) mice. ApoE-/- mice fed a western diet for 10 weeks were administered recombinant mouse IL-9 (rIL-9) or anti-IL-9 neutralizing monoclonal antibody (mAb). Mice treated with rIL-9 developed markedly larger plaques in both the aorta and aortic root. Immunohistochemical studies demonstrated increases in both vascular endothelial adhesion molecule-1 (VCAM-1) expression and the infiltration of inflammatory cells, including T cells and macrophages, in plaques. However, treatment with the anti-IL-9 mAb caused the opposite effect. The administration of rIL-9 did not affect the splenic T cell or peripheral monocyte subsets. Meanwhile, IL-9 induced VCAM-1 expression in MAECs mainly via a STAT3-dependent pathway, consequently increasing monocyte-endothelial adhesion. Moreover, treatment with anti-VCAM-1 mAb partially abrogated the IL-9-induced increase in plaque area. In addition, CD4(+)IL-9(+) T cells and IL-9 were increased in patients with acute coronary syndrome, and the levels of IL-9 in culture supernatants and soluble VCAM-1 (sVCAM-1) in plasma were significantly positively correlated in the enrolled patients. CONCLUSION: Our results demonstrated that IL-9 exerted pro-atherosclerotic effects in ApoE-/- mice at least partially by inducing VCAM-1 expression, which mediated inflammatory cell infiltration into atherosclerotic lesions.

Decreased expression of uteroglobin-related protein 1 in inflamed mouse airways is mediated by IL-9.

Uteroglobin-related protein 1 (UGRP1) is a secretory protein, highly expressed in epithelial cells of airways. Although an involvement of UGRP1 in the pathogenesis of asthma has been suggested, its function in airways remains unclear. In the present study, a relationship between airway inflammation, UGRP1 expression, and interleukin-9 (IL-9), an asthma candidate gene, was evaluated by using a murine model of allergic bronchial asthma. A severe airway inflammation accompanied by airway eosinophilia and elevation of IL-9 in bronchoalveolar lavage (BAL) fluids was observed after ovalbumin (OVA) challenge to OVA-sensitized mice. In this animal model of airway inflammation, lung Ugrp1 mRNA expression was greatly decreased compared with control mice. A significant inverse correlation between lung Ugrp1 mRNA levels and IL-9 levels in BAL fluid was demonstrated by regression analysis (r = 0.616, P = 0.023). Immunohistochemical analysis revealed a distinct localization of UGRP1 in airway epithelial cells of control mice, whereas UGRP1 staining was patchy and faint in inflamed airways. Intranasal administration of IL-9 to naive mice decreased the level of Ugrp1 expression in lungs. These findings suggest that UGRP1 is downregulated in inflamed airways, such as allergic asthmatics, and IL-9 might be an important mediator for modulating UGRP1 expression.

IL-9 protects mice from Gram-negative bacterial shock: suppression of TNF-alpha, IL-12, and IFN-gamma, and induction of IL-10.

IL-9 is a T cell-derived cytokine that, similar to the Th2 cytokines IL-4 and IL-10, has been implicated in the response to parasitic infections, allergy, and inflammatory processes. Because both IL-4 and IL-10 can confer protection to mice from septic shock, we investigated whether IL-9 may also be capable of conferring resistance on recipients of an otherwise lethal challenge with Pseudomonas aeruginosa. Prophylactic injections of rIL-9 appeared to be most effective in preventing the onset of a lethal shock, according to a pattern that was both dose dependent and time dependent. The protective effect of IL-9 was correlated with marked decreases in the production of the inflammatory mediators TNF-alpha, IL-12, and IFN-gamma, as well as the induction of the anti-inflammatory cytokine IL-10. Sustained levels of IL-9-specific transcripts could be detected in the spleens of mice recovering from sublethal P. aeruginosa infection. Therefore, IL-9 may be protective in septic shock via a rather unique mechanism involving a complex modulation of inflammatory and anti-inflammatory mediators.

IL-9 pathway in asthma: new therapeutic targets for allergic inflammatory disorders.

BACKGROUND: Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and bronchial hyperresponsiveness (BHR). The incidence of asthma continues to rise in industrialized countries despite advances in the identification of cellular and molecular mediators that are associated with the disease. Because of its importance in human health, additional research and alternative therapeutic strategies are justified to create more effective treatments for this debilitating disease. OBJECTIVE: Studies use recombinant inbred mice to demonstrate that BHR in mouse models of asthma is associated with a genetic alteration at the IL-9 locus, where IL-9 expression in lung is strongly associated with bronchial responsiveness. We have investigated the ability of intratracheal instilled IL-9 to induce asthmatic-like responses in naive C57BL/6 (B6) mice, which express very low levels of IL-9. METHODS: IL-9 or vehicle was intratracheal instilled in naive B6 mice for 10 days. Mice were analyzed for effects on BHR, lung eosinophilia, and serum total IgE levels. RESULTS: Phenotypic effects of B6 mice instilled with IL-9 were increased eosinophils in the bronchoalveolar lavage and significantly elevated serum total IgE. Moreover, IL-9 was found to induce IL-5Ralpha in vivo and in vitro, suggesting a potential mechanism for the novel actions described for IL-9 on eosinophils. CONCLUSION: Increased levels of IL-9 in the airway of naive B6 mice induced lung eosinophilia and serum total IgE levels, which are 2 clinical features of asthma. These data support a central role for the IL-9 pathway in the complex pathogenesis of allergic inflammation.

Cytokines and thermoregulation: interleukin-9 injected in preoptic area fails to evoke fever in rats.

A number of the members of the family of cytokines including IL-1, IL-2, IL-6, and IL-11 act directly in the brain to induce a febrile response in the rat and other species. The purpose of this study was to examine the effect of interleukin-9 (IL-9) when this cytokine is applied directly to the thermosensitive and pyrogen reactive region of the anterior hypothalamic, preoptic area (AH/POA). In male Sprague-Dawley rats, guide cannulae for microinjection into the AH/POA were implanted stereotaxically, and radio transmitters for monitoring body temperature (Tb) were placed intraperitoneally. Following postoperative recovery, recombinant murine macrophage inflammatory protein (MIP)-1 beta was microinjected in the AH/POA of each rat in a dose of 28 pg/microliters to identify pyrogen reactive sites in the AH/POA. Then recombinant human IL-9 was suspended in pyrogen-free CSF vehicle and microinjected in the same sites in concentrations of 2.4, 24, and 240 U/microliters. In contrast to the pyrexic action of MIP-1 beta, IL-9 failed to elicit a significant alteration in the Tb of the rats at any of the doses tested. IL-9 was also without effect on the intakes of either water or food. These results demonstrate that IL-9 applied to the region of the diencephalon in which other cytokines act to evoke fever may not play a direct role in the thermogenic component underlying the acute phase response. However, as demonstrated in several different cell systems, IL-9 may require a cofactor related to pyrogen for a febrile response to develop.

Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes.

IgE production by normal peripheral blood lymphocytes (PBL) is known to be triggered upon stimulation by interleukin (IL)-4. In the present study we showed that IL-9, another T cell-derived cytokine, markedly potentiated IgE production induced by suboptimal doses of IL-4, whereas no effect of IL-9 was observed in the absence of IL-4. The potentiating effect of IL-9 appeared to be associated with the increased frequency of IgE-producing cells, as revealed by a specific ELISA-spot assay. Under the same experimental conditions, IL-9 also enhanced the IL-4-induced IgG production but did not elicit IgM production. However, IL-9 did not amplify the IL-4-dependent expression of membrane-bound and soluble low affinity receptor for IgE (CD23). IL-4-induced IgE production was also potentiated by IL-6 but not by tumor necrosis factor-alpha and IL-1 beta. The possibility that the activity of IL-9 was mediated by IL-6 released from accessory cells was excluded by the observations that monocyte depletion did not abolish the effect of IL-9 and that IL-9 was still active on fluorescence-assisted cell sorted CD20+ B lymphocytes co-cultured with irradiated murine EL4 cells. In addition, IL-9 was shown to potentiate the IL-4-induced IgG and IgM production by normal human B lymphocytes preactivated with Staphylococcus aureus Cowan strain. Taken together, these data suggest that IL-9 plays a regulatory role in the IL-4-dependent immunoglobulin production.