Interleukin-9 deficiency affects lipopolysaccharide-induced macrophage-related oxidative stress and myocardial cell apoptosis via the Nrf2 pathway both in vivo and in vitro.

Previous studies showed that interleukin-9 (IL-9) is involved in cardiovascular diseases, including hypertension and cardiac fibrosis. This study aimed to investigate the role of IL-9 in lipopolysaccharide (LPS)-induced myocardial cell (MC) apoptosis. Mice were treated with LPS, and IL-9 expression was measured and the results showed that compared with WT mice, LPS-treated mice exhibited increased cardiac Mø-derived IL-9. Additionally, the effects of IL-9 deficiency (IL-9-/-) on macrophage (Mø)-related oxidative stress and MC apoptosis were evaluated, the results showed that IL-9 knockout significantly exacerbated cardiac dysfunction, inhibited Nrf2 nuclear transfer, promoted an imbalance in M1 and M2 Møs, and exacerbated oxidative stress and MC apoptosis in LPS-treated mice. Treatment with ML385, a specific nuclear factor erythroid-2 related factor 2 (Nrf2) pathway inhibitor significantly alleviated the above effects in LPS-treated IL-9-/- mice. Bone marrow-derived Møs from wild-type (WT) mice and IL-9-/- mice were treated with LPS, and the differentiation and oxidative stress levels of Møs were measured. The effect of Mø differentiation on mouse MC apoptosis was also analyzed in vitro. The results showed that LPS-induced M1 Mø/M2 Mø imbalance and Mø-related oxidative stress were alleviated by IL-9 knockout but were exacerbated by ML385 treatment. The protective effects of IL-9 deficiency on the MC apoptosis mediated by LPS-treated Møs were reversed by ML-385. Our results suggest that deletion of IL-9 decreased the nuclear translocation of Nrf2 in Møs, which further aggravated Mø-related oxidative stress and MC apoptosis. IL-9 may be a target for the prevention of LPS-induced cardiac injury.

IL-9 Deficiency Promotes Pulmonary Th17 Response in Murine Model of Pneumocystis Infection.

Introduction: Pneumocystis pneumonia (PCP) remains a severe complication with high mortality in immunocompromised patients. It has been well accepted that CD4+ T cells play a major role in controlling Pneumocystis infection. Th9 cells were the main source of IL-9 with multifaced roles depending on specific diseases. It is unclear whether IL-9/Th9 contributes to the immune response against PCP. The current study aims to explore the role of IL-9 and the effect of IL-9 on Th17 cells in murine model of PCP. Materials and methods: Mice were intratracheally injected with 1 × 106Pneumocystis organisms to establish the murine model of Pneumocystis infection. Pneumocystis burden was detected by TaqMan real-time PCR. Using IL-9-deficient (IL-9-/-) mice, flow cytometry, real-time PCR and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the immune function related to Th17 response in defense against Pneumocystis infection. Results: Reduced Pneumocystis burden was observed in lungs in IL-9-/- mice compared with WT mice at 3-week postinfection. IL-9-/-mice exhibited stronger Th17 immune responses than WT PCP mice through flow cytometer and real-time PCR. ELISA revealed higher levels of IL-17 and IL-23 in bronchoalveolar lavage fluid from IL-9-/- mice than WT mice. And IL-9 deficiency promoted Th17 differentiation from CD4+ naive T cells. IL-17A neutralization increased Pneumocystis burden in IL-9-/- mice. Conclusion: Although similar basic clearance of Pneumocystis organisms was achieved in both WT and IL-9-/- PCP mice, IL-9 deficiency could lower Pneumocystis organism burden and promote pulmonary Th17 cells response in the early stage of infection.

Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.

IL-9 is important for T-cell activation and differentiation in autoimmune inflammation of the central nervous system.

Experimental autoimmune encephalomyelitis (EAE) is generally believed to be an autoimmune disease of the central nervous system (CNS) caused by myelin-specific Th1 and/or Th17 effector cells. The underlying cellular and molecular mechanisms, however, are not fully understood. Using mice deficient in IL-9 (IL-9(-/-) ), we showed that IL-9 plays a critical role in EAE. Specifically, IL-9(-/-) mice developed significantly less severe EAE than their WT counterparts following both immunization with myelin proteolipid protein (PLP)(180-199) peptide in the presence of Complete Freund's Adjuvant (CFA), and adoptive transfer of PLP(180-199) peptide-specific effector T cells from WT littermates. EAE-resistant IL-9(-/-) mice exhibited considerably fewer infiltrating immune cells in the CNS, with lower levels of IL-17 and IFN-γ expression, than their WT littermates. Further studies revealed that null mutation of the IL-9 gene resulted in significantly lower levels of PLP(180-199) peptide-specific IL-17 and IFN-γ production. Moreover, IL-9(-/-) memory/activated T cells exhibited decreased C-C chemokine receptors (CCR)2, CCR5, and CCR6 expression. Interestingly, IL-10 was significantly increased in IL-9(-/-) mice compared with WT littermates. Importantly, we found that IL-9-mediated Th17-cell differentiation triggers complex STAT signaling pathways.

IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression.

Both mast cells (MCs) and regulatory T cells (Tregs) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MCs and Tregs have been shown to play a protective role. Transfer of wild-type (wt) Tregs into wt recipients almost completely prevents development of NTS and leads to a profound increase of MCs in the renal draining lymph nodes (LNs). By contrast, transfer of wt Tregs into animals deficient in MCs, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a mAb in mice receiving Tregs abrogated protection from NTS. Moreover, transfer of IL-9-deficient Tregs also failed to protect from NTS. In the absence of Treg-derived IL-9, MCs fail to accumulate in the LNs, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Tregs. In summary, to our knowledge, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining LNs.

Chronic allergen challenge induces bronchial mast cell accumulation in BALB/c but not C57BL/6 mice and is independent of IL-9.

As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (<90 microm), medium (90-155 microm), or large (>150 microm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.

IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity.

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9-deficient mice fail to develop experimental oral antigen-induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9-mediated mast cell responses have an important role in food allergy.

IL-13 mediates in vivo IL-9 activities on lung epithelial cells but not on hematopoietic cells.

Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.

IL-9 is a susceptibility factor in Leishmania major infection by promoting detrimental Th2/type 2 responses.

IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.

The absence of interleukin 9 does not affect the development of allergen-induced pulmonary inflammation nor airway hyperreactivity.

Interleukin (IL)-9 is a pleiotropic cytokine secreted by T helper (Th)2 cells and has been proposed as a candidate gene for asthma and allergy. We have used mice genetically deficient in IL-9 to determine the role of this cytokine in the pathophysiologic features of the allergic pulmonary response-airway hyperreactivity (AHR) and eosinophilia. We have demonstrated that IL-9 is not required for the development of a robust Th2 response to allergen in sensitized mice. IL-9 knockout mice developed a similar degree of eosinophilic inflammation and AHR to their wild-type littermates. Goblet cell hyperplasia and immunoglobulin (Ig) E production were also unaffected by the lack of IL-9. Moreover, levels of bronchoalveolar lavage (BAL) IL-4, IL-5, and IL-13 were comparable between wild-type and knockout mice. These findings indicate that IL-9 is not obligatory for the development of eosinophilia and AHR, and imply that other Th2 cytokines can act in a compensatory fashion.

Th2 cytokines and asthma. Interleukin-9 as a therapeutic target for asthma.

Asthma is a complex heritable inflammatory disorder of the airways in which the development of clinical disease depends on environmental exposure. It has been well established that T helper type 2 (TH2) lymphocytes and their cytokines have an important role in allergic asthma. Interleukin (IL)-9, a member of the TH2 cytokine family, has recently been implicated as an essential factor in determining mucosal immunity and susceptibility to atopic asthma. In this review we examine the critical experiments and observations that support this hypothesis. We also discuss these results in comparison with the experiments supporting the involvement of other T H2 cytokines such as IL-4, IL-5 and IL-13.

IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development.

Interleukin-9 is a cytokine produced by Th2 cells and is a candidate gene for asthma and atopy. We have generated IL-9-deficient mice to delineate the specific roles of IL-9 in Th2 responses. Using a pulmonary granuloma model, we have demonstrated a distinct requirement for IL-9 in the rapid and robust generation of pulmonary goblet cell hyperplasia and mastocytosis in response to lung challenge. In contrast, eosinophilia and granuloma formation were not affected. IL-9 was not required for T cell development or differentiation, the generation of naive or antigen-driven antibody responses, or the expulsion of the intestinal parasitic nematode Nippostrongylus brasiliensis. Thus, deletion of IL-9 manifests as a highly defined phenotype in Th2 responses modulating mucus production and mast cell proliferation.