A critical role for C5L2 in the pathogenesis of experimental allergic asthma

The complement fragment C5a plays dual roles in the development of experimental allergic asthma. It protects from pulmonary allergy by a regulatory effect on dendritic cells during allergen sensitization, but is proallergic during the effector phase. C5a can bind to two distinct receptors (i.e., C5a receptor and C5a receptor-like 2 [C5L2]). The functional role of C5L2 in vivo remains enigmatic. In this study, we show in two models of OVA- and house dust mite (HDM)-induced experimental allergic asthma that C5L2-deficient mice are protected from the development of airway hyperresponsiveness, Th2 cytokine production, eosinophilic airway inflammation, serum IgE, or mucus production. Surprisingly, HDM-induced experimental asthma in C5L2-deficient mice was associated with increased pulmonary IL-17A production and increased airway neutrophil numbers. To directly assess the role of C5L2 on myeloid dendritic cells (mDCs) during allergen sensitization, we performed single or repeated adoptive transfers of C5L2-deficient mDCs into wild-type mice. HDM-pulsed C5L2-deficient mDCs induced strong Th2 cytokine production, which was associated with marked IFN-γ and IL-17A production, decreased eosinophil numbers, and reduced IgE production as compared with HDM-pulsed mDCs from wild-type mice. HDM stimulation of C5L2(-/-) mDCs in vitro resulted in production of Th17-promoting cytokine IL-23, which was absent in wild-type mDCs. Our findings suggest that C5L2 acts at the mDC/T cell interface to control the development of Th1 and Th17 cells in response to airway HDM exposure. Furthermore, it drives Th2 immune responses independent of mDCs, suggesting a complex role for C5L2 in the development of experimental allergic asthma.     

IL-9 promotes Th17 cell migration into the central nervous system via CC chemokine ligand-20 produced by astrocytes

Newly discovered IL-9-producing helper T cells (Th9) reportedly exert both aggravating and suppressive roles on experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, it is still unclear whether Th9 is a distinct Th cell subset and how IL-9 functions in the CNS. In this study, we show that IL-9 is produced by naive CD4(+) T cells that were stimulated with anti-CD3 and anti-CD28 Abs under the conditions of Th2-, inducible regulatory T cell-, Th17-, and Th9-polarizing conditions and that IL-9 production is significantly suppressed in the absence of IL-4, suggesting that IL-4 is critical for the induction of IL-9 by each producing cell. The IL-9 receptor complex, IL-9R and IL-2Rγ, is constitutively expressed on astrocytes. IL-9 induces astrocytes to produce CCL-20 but not other chemokines, including CCL-2, CCL-3, and CXCL-2 by astrocytes. The conditioned medium of IL-9-stimulated astrocytes induces Th17 cell migration in vitro, which is cancelled by adding anti-CCL-20 neutralizing Abs. Treating with anti-IL-9 neutralizing Abs attenuates experimental autoimmune encephalomyelitis, decreases the number of infiltrating Th17 cells, and reduces CCL-20 expression in astrocytes. These results suggest that IL-9 is produced by several Th cell subsets in the presence of IL-4 and induces CCL-20 production by astrocytes to induce the migration of Th17 cells into the CNS.     

Interleukin-9 stimulates the production of interleukin-5 in CD4+ T cells

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.     

Lymphomagenesis, hydronephrosis, and autoantibodies result from dysregulation of IL-9 and are differentially dependent on Th2 cytokines

Interleukin-9 is an immunoregulatory cytokine implicated in the development of asthma and allergy. To investigate the role of IL-9 in vivo, we have generated transgenic mice in which IL-9 is expressed from its own promoter. Strikingly, overexpression of IL-9 resulted in premature mortality associated with a complex phenotype characterized by the development of autoantibodies, hydronephrosis, and T cell lymphoma. By intercrossing IL-9 transgenic mice with a panel of Th2 cytokine-deficient mice, we demonstrate that these disorders represent distinct phenotypes that can be dissociated by their differential dependence on Th2 cytokines. Autoantibody production was ablated in IL-9 transgenic animals with a combined absence of IL-4, IL-5, and IL-13, coincident with a reduction in peritoneal B-1 cells. Hydronephrosis arose in 75% of IL-9 transgenic animals and was dependent on the presence of IL-4 and IL-13. In contrast, T cell lymphomas developed independently of the other Th2 cytokines, with the generation of rapidly proliferating CD8(+) or CD4(+)CD8(+) T cell clones that arose in the thymus before infiltrating both lymphoid and nonlymphoid tissues. Our data highlight potentially important new roles for IL-9, through its regulation of downstream Th2 effector cytokines, in autoantibody production and in hydronephrosis.     

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.     

Antigen-specific Th9 cells exhibit uniqueness in their kinetics of cytokine production and short retention at the inflammatory site

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-β and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the "natural mode" (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9-producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.     

A plant-based allergy vaccine suppresses experimental asthma via an IFN-gamma and CD4+CD45RBlow T cell-dependent mechanism

Allergic asthma is currently considered a chronic airway inflammatory disorder associated with the presence of activated CD4(+) Th2-type lymphocytes, eosinophils, and mast cells. Interestingly, therapeutic strategies based on immune deviation and suppression have been shown to successfully attenuate the development of the asthma phenotype. In this investigation, we have for the first time used a genetically modified (GM) plant, narrow leaf lupin (Lupinus angustifolius L.), expressing a gene for a potential allergen (sunflower seed albumin) (SSA-lupin) to examine whether a GM plant/food-based vaccine strategy can be used to suppress the development of experimental asthma. We show that oral consumption of SSA-lupin promoted the induction of an Ag-specific IgG2a Ab response. Furthermore, we demonstrate that the plant-based vaccine attenuated the induction of delayed-type hypersensitivity responses and pathological features of experimental asthma (mucus hypersecretion, eosinophilic inflammation, and enhanced bronchial reactivity (airways hyperreactivity). The suppression of experimental asthma by SSA-lupin was associated with the production of CD4(+) T cell-derived IFN-gamma and IL-10. Furthermore, we show that the specific inhibition of experimental asthma was mediated via CD4(+)CD45RB(low) regulatory T cells and IFN-gamma. Thus, our data demonstrate that a GM plant-based vaccine can promote a protective immune response and attenuate experimental asthma, suggesting that plant-based vaccines may be potentially therapeutic for the protection against allergic diseases.     

V alpha 24-invariant NKT cells from patients with allergic asthma express CCR9 at high frequency and induce Th2 bias of CD3+ T cells upon CD226 engagement

We have demonstrated that Valpha24(+)Vbeta11(+) invariant (Valpha24(+)i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Valpha24(+)i NKT cells but not the normal cells. A large number of CCR9-positive Valpha24(+)i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Valpha24(+)i NKT cells, themselves Th1 biased, induce CD3(+) T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Valpha24(+)i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Valpha24(+)i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Valpha24(+)i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Valpha24(+)i NKT cells to induce Th2-cytokine production by CD3(+) T cells, indicating that CD226 engagement is necessary for Valpha24(+)i NKT cells to induce Th2 bias of CD3(+) T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Valpha24(+)i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Valpha24(+)i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Valpha24(+)i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.     

IL-1 serves as a secondary signal for IL-9 expression.

IL-9 is produced in vitro by activated CD4+ T cell lines of the Th2 subtype and by naive CD4+ T cells. Here we show that T cell lines stimulated with Con A in the presence of accessory cells (AC) such as irradiated spleen cells or bone marrow-derived macrophages produced substantially more IL-9 than T cells stimulated with Con A alone. These data suggest that AC influence the production of IL-9 through accessory signals that result in an at least 10-fold increase of IL-9 secretion by the respective T cells. Addition of IL-1 to T cells activated by Con A, PHA, or anti-CD3 antibodies revealed that this monokine was responsible for the potentiation of IL-9 production. This finding was confirmed by applying anti-IL-1 antibodies. The production of other lymphokines, namely, IL-3, IL-4, and IL-6, by activated T cells was not or only marginally enhanced in the presence of AC or IL-1, thus indicating that the synthesis of IL-9 is regulated differently from that of other Th2-derived lymphokines. Furthermore, it was demonstrated by Northern blot analysis that IL-1 increases IL-9 expression at the pretranslational level. Because IL-1 alone failed to induce the production of IL-9 by T cells, this monokine acts as a costimulator in combination with a T cell receptor-mediated signal.     

Chlamydial Lung Infection Induces Transient IL-9 Production Which Is Redundant for Host Defense against Primary Infection

IL-9/Th9 responses are recently found to be important for innate and adaptive immunity particularly in parasitic infections. To date, the study on the role of IL-9 in bacterial infections is limited and the reported data are contradictory. One reported function of IL-9/Th9 is to modulate Th1/Th17 responses. Since our and others’ previous work has shown a critical role of Th1 and Th17 cells in host defense against chlamydial lung infection, we here examined the role of IL-9 responses in Chlamydia muridarum (Cm) lung infection, particularly its effect on Th1 and Th17 responses and outcome infection. Our data showed quick but transient IL-9 production in the lung following infection, peaking at day 3 and back to baseline around day 7. CD4+ T cell was the major source of IL-9 production in the lung infection. Blockade of endogenous IL-9 using neutralizing antibody failed to change Interferon-γ (IFN-γ) and IL-17 production by cultured spleen mononuclear cells isolated from Cm infected mice. Similarly, in vivo neutralization of IL-9 failed to show significant effect on T cell (Th1 and Th17) and antibody responses (IgA, IgG1 and IgG2a). Consistently, the neutralization of IL-9 had no significant effect on disease process, including body weight change, bacterial burden and histopathological score. The data suggest that IL-9 production following chlamydial lung infection is redundant for host defense against the intracellular bacteria.     

IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.     

In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (alpha or beta) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.     

Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively. A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type 2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble ST2 directly bound to IL-33 and suppressed activation of NF-kappaB in EL-4 cells stably expressing ST2L, suggesting that the complex of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates the biological activity of IL-33 in allergic airway inflammation.     

Computational and experimental analysis reveals a requirement for eosinophil-derived IL-13 for the development of allergic airway responses in C57BL/6 mice

Eosinophils are found in the lungs of humans with allergic asthma, as well as in the lungs of animals in models of this disease. Increasing evidence suggests that these cells are integral to the development of allergic asthma in C57BL/6 mice. However, the specific function of eosinophils that is required for this event is not known. In this study, we experimentally validate a dynamic computational model and perform follow-up experimental observations to determine the mechanism of eosinophil modulation of T cell recruitment to the lung during development of allergic asthma. We find that eosinophils deficient in IL-13 were unable to rescue airway hyperresponsiveness, T cell recruitment to the lungs, and Th2 cytokine/chemokine production in ΔdblGATA eosinophil-deficient mice, even if Th2 cells were present. However, eosinophil-derived IL-13 alone was unable to rescue allergic asthma responses in the absence of competence of other IL-13-producing cells. We further computationally investigate the role of other cell types in the production of IL-13, which led to the various predictions including early and late pulses of IL-13 during airway hyperresponsiveness. These experiments suggest that eosinophils and T cells have an interdependent relationship, centered on IL-13, which regulates T cell recruitment to the lung and development of allergic asthma.     

The TLR7 ligand 9-benzyl-2-butoxy-8-hydroxy adenine inhibits IL-17 response by eliciting IL-10 and IL-10-inducing cytokines

This study evaluates the ability of a novel TLR7 ligand (9-benzyl-2-butoxy-8-hydroxy adenine, called SA-2) to affect IL-17 response. The SA-2 activity on the expression of IL-17A and IL-17-related molecules was evaluated in acute and chronic models of asthma as well as in in vivo and in vitro α-galactosyl ceramide (α-GalCer)-driven systems. SA-2 prepriming reduced neutrophils in bronchoalveolar lavage fluid and decreased methacoline-induced airway hyperresponsiveness in murine asthma models. These results were associated with the reduction of IL-17A (and type 2 cytokines) as well as of molecules favoring Th17 (and Th2) development in lung tissue. The IL-17A production in response to α-GalCer by spleen mononuclear cells was inhibited in vitro by the presence of SA-2. Reduced IL-17A (as well as IFN-γ and IL-13) serum levels in mice treated with α-GalCer plus SA-2 were also observed. The in vitro results indicated that IL-10 produced by B cells and IL-10-promoting molecules such as IFN-α and IL-27 by dendritic cells are the major player for SA-2-driven IL-17A (and also IFN-γ and IL-13) inhibition. The in vivo experiments with anti-cytokine receptor Abs provided evidence of an early IL-17A inhibition essentially due to IL-10 produced by resident peritoneal cells and of a delayed IL-17A inhibition sustained by IFN-α and IL-27, which in turn drive effector T cells to IL-10 production. These findings suggest that such TLR7 agonist downregulating Th17 (as well as Th2) response has to be considered a valid candidate for novel vaccine formulations in allergy.