Jinna Yang, Yuyang Zhang, Linglong Yang, Huimin Wen, Xiaoxue Jiang, Lu Dong

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, P.R. China

Application of transgenic mice to the studies of inflammatory factors associated with asthma

Jinna Yang, Yuyang Zhang＊, Linglong Yang, Huimin Wen, Xiaoxue Jiang, Lu Dong

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, P.R. China

Asthma is a chronic inflammatory disorder of airways characterized by prominent inflammatory cells infiltrated. And these inflammatory cells can secrete a series of inflammatory cytokines which are critical in the research of the occurrence and development of asthma. In vivo animal models can offer valuable information on several aspects of asthma pathogenesis and treatment. Transgenic mice, helping us efficiently and accurately define the function of specific genes in lungs, can be made animal models needed in evaluating the potential contribution to the pathogenesis and progression made by asthma inflammatory cells and cytokines. In this review, we will discuss the application of transgenic mice to the research on the onset and development of asthma in recent years, especially the inflammatory factors involved in them. And transgenic mice will play a more important role in the study of anti-asthma drugs than ever before.

transgenic mice; asthma; inflammation; cytokines

1 Introduction

Asthma is characterized by chronic inflammation occurred in airways where overabundance of eosinophils, mast cells, T lymphocytes activated, neutrophils and epithelial cells exists. These inflammatory cells release mediators, such as interleukins (IL), leukotrienes (LT), transforming growth factors (TGF) and a variety of chemokines. The mediators trigger airway inflammation and promote the development of airway hyper-responsiveness (AHR). Excessive mucus secretes from airways due to the goblet-cell hyperplasia and thickened airway wall, which leads to clinical symptoms related to the disease, like reversible airflow obstruction, repeated episodes of wheezing, dyspnea or cough [1].

Cytokines associated with asthma generally mean lymphokines secreted by T cells that regulate immune responses, pro-inflammatory cytokines that amplify and maintain inflammatory process, antiinflammatory cytokines that negatively modulate inflammatory responses, chemokines that are chemotactic for inflammatory cells and growth factors that promote cell survival and result in structural changes in airways [2]. The inflammatory cytokines are important elements in orchestrating thechronic inflammation of asthma by recruiting and activating multiple inflammatory cells in respiratory tracts as well as promoting them survival [3, 4].

Transgenic (Tg) mice are widely used in biomedical research for investigating the phenotypic effects of expressed transgenes. In this case, promoters are often utilized to control the transgene expression in the specific tissues, or to study the regulation of gene expression in an intact animal. Therefore, the specific patterns of the expression in various tissues are made clear with potential control sequence elements. The given genes expressed in Tg mice and two RNAs, i.e. encoding antisense one or short interfering one, can successfully inhibit the expression of endogenous genes in the mice [5, 6]. Genetic studies have linked asthma, atopy, and bronchial hyper-responsiveness to human chromosome 5q31-q33 that contains several genes involved in the allergic immune response. Interstrain genetic and phenotypic differences are the genetic basis of the diseases. Tg mice, of which cytokines are polarized, lead to better understanding of the function of specific genes in lungs. Therefore, as important animal models, they are used to observe the potential of inflammatory cells and cytokines in the pathogenesis and progression of lung diseases and to analyze the effect of a single cytokine on a single cell type with asthma. Furthermore, these results can guide translational studies that show the relevance of some of these gene expression changes in people with asthma.

During the past few years, many Tg mouse lines have been established for research on asthma [7, 8]. The researches on the Tg models give us new insights into what is happening to the pathogenesis of asthma and its potential treatment. The present review discusses the application of Tg mice to the studies of asthmatic occurrence and development in recent years, especially to those of the inflammatory factors involved in them.

2 Transgenic mice used in studies of proinflammatory cytokines in asthma

Asthma is generally caused by viral infections or other allergens exposures. In this case, the cytokines responsible to T helper type 2 (Th2), which derive from airway epithelial cells, including IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), a newly discovered cytokine, are critical to initiate Th2 inflammatory responses and airway lesions. The cytokines take action through dendritic cells (DCs) and then drive Th2 cell to differentiate and release IL-4, IL-5, IL-9 and IL-13, etc. (Fig. 1). Moreover, the cytokines have an effect on inflammatory cells such as eosinophils, mast cells, neutrophils and basophils, to produce the differentiation of B lymphocytes and the generation of immunoglobulin (Ig) E, and enhance airway inflammation and AHR [9, 10]. However, in recent years, it has been recognized that Th1/Th2 imbalance does not fully explain the etiology of asthma. For instance, reversing the imbalance between Th1 and Th2 does not completely control asthmatic symptoms in humans. Some studies have suggested that other CD4+T cell subsets may play a role in asthma, including Th1 cells, Th17 cells and regulatory T cells (Treg). The balance between Th17 and Treg may do an important job of developing asthma. Th1 subset stimulates cell-mediated immune responses, especially those to pathogens, mainly via the production of IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, while Th2 is characterized by producing IL-4, IL-5, IL-9 and IL-13 that are the main factors of asthma pathogenesis (Fig. 1). An imbalance between Th1 and Th2 cells may start allergic asthma, while an imbalance in Th17/ Treg cells is associated with its deterioration. It is described in the study conducted by Shi Yuheng [11] that the imbalance between Th1 and Th2 as well as that between Th17 and Treg are found in patientswith allergic asthma. The frequency of blood Th2 cells and IL-4 levels in plasma and the supernatant of peripheral blood mononuclear cells cultured were increased, and that of Th17 cells and IL-17 levels in plasma and culture supernatant were raised in all patients with allergic asthma, whereas the frequency of CD4+CD25+Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. In addition, 4-1BB ligand (4-1BBL), which can control the balance between Th17 and Treg, were reduced in patients with allergic asthma compared with the control subjects [12].

Th2 cells are involved in asthma by releasing various cytokines. IL-4 and IL-13 are important elements in the induction and regulation of allergic asthma through their effects on production of Ig E and mucous secretion and AHR. IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF) promote the differentiation and migration of eosinophils [3]. Large genomic segments containing IL-4, IL-13 and IL-5 genes were implanted into bacterial artificial chromosome (BAC) Tg mice, therefore the Th2 cytokines expressed over in the relevant tissues of the mice. Then these mice were used to investigate the effects of the Th2 cytokines on the development in asthma. The results show that too much expression of the cytokines enhances the airway inflammation caused by environmental allergens. The infiltration of inflammatory cells, the hypertrophy of airway epithelial cells and the high level of serum IgE are the hallmarks of atopic diseases. All these provide additional evidence to support the critical role of Th2 cytokines in the pathogenesis of asthma [13].

IL-4, a typical cytokine produced by Th2 cells, mast cells and eosinophils, is necessary for the differentiation and expansion of Th2, and can suppress Th1 development. It increases the expression of major histocompatibility complex class II (MHCII) molecules, up-regulates the expression of FcoR on the surface of B cells and promotes the differentiation of Th2 cells and type 2 cytokine responses. In addition, IL-4 is significant for antibody responses switched by IgG1 and IgE isotypes, which enhances the immunoreaction mediated by IgE [14]. As the major stimulus of Th2 cell development, IL-4 is also adjusted by factor c-Maf. It combines with its receptor to promote the uncommitted naive CD4+T cells to differentiate into Th2 and regulates the secretion of Th2 cytokines, which directly or indirectly induce mast cells and eosinophils to degranulate and finally lead to the enhanced AHR and inflammation [15].

IL-5 is produced by Th2 and mast cells after repeated allergeic challenges. It integrates with IL-5R to induce terminal differentiation of activated B cells into the cells with antibody, and then enhances the proliferation and differentiation of eosinophil precursors into mature eosinophils as well as the release of eosinophils into blood. The research of Kouro and Takatsu [16] demonstrated that lacking a functional gene for IL-5 or the IL-5 receptor alpha chain (IL-5Rα) displayed a number of developmental and functional impairments in B-cells and eosinophil lineages. IL-5-Tg mice were taken to investigate the effect of airway eosinophilia on respiratory tract. After sensitization and challenge with ovalbumin (OVA), the Tg mice had a marked eosinophil increase in airways which showed a lower reactivity to methacholine. Immunohistochemical analysis of lungs revealed a prominent peribronchial infiltration of eosinophils. The anti-IL-5 treatment decreased the levels of both eosinophil and TGF-β1 in bronchoalveolar lavage fluids (BALF) and increased AHR as well [17].

IL-9, a pleiotropic cytokine also produced by the Th2 subset, is in connection with the function of mast cells, B lymphocytes, eosinophils and airway epithelial cells. IL-9-Tg mice were used to identify the effect of IL-9 on airway inflammation in asthma. IL-9 transgene induces lymphocytic and eosinophilic infiltration in lungs, airway epithelialcell hypertrophy with mucous production, mast cell hyperplasia and expression of various cytokines in the tissues, like IL-4, IL-5 and IL-13. The data [18] showed an obviously growing number of mast cells in the airway epithelium and markedly increasing AHR to some extent after methacholine treatment, which finally resulted in airway inflammation and mast cell hyperplasia, etc. Moreover, there was an increased IL-13 and IL-5 production in innate lymphoid cells (ILC) upon IL-9 stimulation, which suggested that the IL-9-mediated airway inflammation might be the result of enhanced IL-13 and IL-5 expression from ILC [19].

IL-13 is responsible for the development in the early stage of asthma and allergic reaction through promoting proliferation and differentiation of B cells and increasing IgE synthesis, which leads to further amplified antigen-specific inflammatory responses. The trait of asthma is AHR, and its response is often marked by Th2-mediated eosinophilic airway inflammation with mast cell infiltration in airway smooth muscle (ASM) bundles. IL-13 overexpression can be seen in many patients with severe asthma. IL-13 multiplies in sputum and a lot of IL-13+ cells exist under the bronchial mucus and in ASM bundles. The expression of the cytokine is related to asthma control and the intensity of eosinophilic inflammation [20]. Fallon et al [21] considered that IL-13-Tg mice were highly predisposed to fatal anaphylaxis induced by allergen sensitization. An elevated serum level of allergenspecific IgE and a promoted mast cell degranulation and histamine release were observed, and finally there were some induced typical phenotypic changes, including airway remodeling, goblet cell hyperplasia, and eosinophil infiltration with crystal deposition. Tg mice were also used to elucidate the action of eosinophils in chronic IL-13-mediated pathology. IL-13 transgene over-expression in lungs induces the features of chronic inflammatory disorders, containing an eosinophil-rich inflammatory cell infiltration, AHR, and remodeling of the airway. Patricia [22] reported that there was a profound reduction in IL-13-induced eosinophil recruitment into the pulmonary lumen without eotaxin-2 or CC chemokine receptor (CCR) 3, while there was a fourfold increase in it in the absence of eotaxin-1; IL-13 Tg mice deficient in CCR3 had a 98% reduction in lung eosinophils, which correlated with decrease in IL-13-induced mucous cell metaplasia and collagen deposition.

IL-25, also known as IL-17E because it is a member of the IL-17 cytokine family, is produced by the activated Th2 cells, mast cells and epithelial cells. It can act on other cells to multiply allergic response [23]. In the early stage of T cell activation, IL-25 potentiates the expression of the nuclear factor of activated T cells c1 (NFATc1) and Jun B on transcription level, which probably leads to the results of an increased level of initial IL-4 production, up-regulation of GATA-3 expression, an enhanced Th2 cell differentiation and the production of Th2 cytokines, as well as the augmentation of Th2 central memory cells [24]. Both murine (m) IL-25-Tg mice [25] and human (h) IL-25-Tg mice [26] were used to identify the action of IL-25 in vivo. Exposure to the IL-25-Tg mice resulted in a Th2-biased immune response, characterized by an increase of eosinophilia, serum IgE and IgG1, and a Th2 cytokine profile including the elevated level of IL-4, IL-13 and IL-5 in serum [25]. Another study showed that pulmonary inflammation in mice was significantly reduced by soluble IL-25 receptor (IL-25R) and anti-IL-25 antibodies (IL-25mAb). The combination of IL-25 and IL-25R was inhibited by IL-25mAb [27]. Collectively, IL-25 represents a prospective target of therapy for asthma. Further studies are required to determine the function of IL-25 inhibitors and their mechanisms.

IL-33, also named IL-1F11 or NF-HEV, is a nuclear factor derived from high endothelial venules. It is a newly described member of IL-1 family andcan not only mediate lots of biological effects via ST2 receptor which is selectively expressed on Th2 cell and mast cell, but also activate nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs). In addition, it drives the production of Th2-associated cytokines from the polarized Th2 cells in vitro [28, 29]. The function of IL-33 in asthma was examined with IL-33-Tg mice. Histological analysis showed there were massive airway inflammations with the infiltration of eosinophils around bronchi and small blood vessels, the hyperplasia of goblet cells and the accumulation of mucus in pulmonary tissues, the overproduction of IL-5, IL-8 and IL-13 in BALF [30]. The investigation of the effect of IL-33 on DC functions in vitro and in vivo was conducted through the mouse models with allergic lung inflammation induced with OVA. The research [31] stated that the factor directly increased the activity of DCs, stimulated the expression of costimulatory molecules as well as induced DC recruitment and activation in lungs. Moreover, the airway inflammation was reduced in ST2-deficient mice in which DCs activation and migration could not be achieved, in other words, IL-33 activated DCs when antigen was present, which made Th2-type response to the allergic infammation of lungs. In the asthmatic subjects of varying severity, there was an expanding expression of IL-33 in bronchial biopsies, which was consistent with the extent of the disease [32]. According to what is stated above, IL-33, as a proinflammatory cytokine associated with Th2 cell, is a critical factor of asthmatic inflammation. The interaction of IL-33 and its membrane receptors on target cells might be inhibited by the binding of IL-33 and soluble ST2, and thus the activation of NF-κB was suppressed, which probably relieved the airway inflammation [33].

TSLP, another newly found cytokine like IL-7, is mainly expressed in the epithelial cells of the lung, intestine and skin and involved in the development and differentiation of B lymphocytes and the proliferation of T lymphocytes, specifically crucial in regulating the activation, differentiation, maturation and migration of DCs [34]. TSLP plays its role in concert with two other cytokines IL-25 and IL-33 described above. Their final effects involve in causing Th2-type inflammation, inducing DCs to make the expression of MHCI, MHCII and co-stimulatory molecules including CD40, CD80 and CD86 up-regulated, and promoting the differentiation of naive CD4+T cells into Th2 cells which release IL-4, IL-5 and IL-13 to participate in the pathogenesis of asthma [35]. TSLP could promote the development of asthma, which was identified in TSLP-Tg mice. The results showed that TSLP was up-regulated in the lungs of the mice with OVA-induced allergic asthma. They presented airway inflammation, AHR, remodeling airway, over mucous secretion, up-regulation of CCL17, a Th2 cell-activating chemokine, and massive eosinophilic infiltration. In contrast, TSLP+/-and TSLP-/-mice demonstrated a considerable decrease in severity when sensitized and challenged with OVA and their inflammatory cells infiltration was only about 10% of wild mice [36]. TSLP antibody may be potential in inhibiting DCs activation, reducing Th2 cells secretion and alleviating inflammation of asthma. In addition, activated pulmonary Treg expressed TSLP-R and responded to TSLP-mediated activation of STAT5. TSLP directly and selectively impaired IL-10 production of Treg and inhibited their suppressive activity. In human with allergic asthma, pulmonary Treg exhibited a significant decrease in suppressive activity and IL-10 production compared to the healthy control and the counterparts with nonallergic asthma [37]. These functional alterations were associated with elevated TSLP expression in BALF of the subjects with allergic asthma. The BALF, furthermore, could inhibit IL-10 produced by the pulmonary Treg of the healthy control in a TSLP-dependent manner.

3 Transgenic mice used in studies of antiinflammatory cytokines involved in asthma

T-regulatory cells (Tregs), potent immunomodulators in the treatment of allergic asthma, can be classified into two general subsets: thymusderived naturally occurring cells (CD4+CD25+Treg) and those induced in the periphery tissues (induced Tregs, iTreg). Reduced AHR, the recruitment of eosinophils, and Th2 cytokine expression in lungs could be seen after allergic challenge in OVA-sensitized mice which were transferred OVA peptide-specific CD4+CD25+Tregs [38]. Green fluorescent protein (GFP)-Tg mice were the subjects used to investigate the action of Tregs in asthma, and GFP-labeled NTregs and iTregs were adoptively transferred into the mice sensitized and challenged by cockroach. The results demonstrated there was much expression of programmed death (PD)-1 in the lungs of the Tg mice, which was an immunoinhibitory receptor expressed in activated T cells, B cells, and myeloid cells, while the levels of IL-4, IL-5 and IL-13 were significantly reduced in BALF. The Tregs, differentiated into CD4+type 1 cells which could yield IL-10, were likely to exert their suppressive activity by increasing the levels of TGF-β, IL-10, IFN-γ and PD-1 and in turn to reverse AHR and airway inflammation in asthma [39].

TGF-β1, a pleiotropic regulatory cytokine with the properties of anti-inflammation and immunosuppression, is produced by various activated cells such as macrophages, epithelial cells, fibroblasts and eosinophils. It is a key factor in the maintenance of immunological homeostasis. The cytokine regulates allergic inflammation and airway remodeling through blocking the differentiation of Th1 and Th2 cells, which are responsible for inhibiting autoimmune responses mediated by Th1 cells and allergic responses mediated by Th2 cells [40, 41]. In addition, it is not only linked to the growth and transformation of cells, but also connected with the repair and regulation of tissues in inflammatory and immune responses. Moreover, TGF-β1 plays an important role in the differentiation of iTregs and Th cells which can produce IL-17, makes Th2 cells lose their characters, increases IL-9 secretion and also directly forces Th 9 cells to differentiate when combined with IL-4 [42]. The results of Scherf's research [43] showed that TGF-β1 protein levels in TGF-β1+/-mice were only about 30% that of wild mice and the reduced expression was accompanied by a strikingly increased eosinophilic inflammation and mucus secretion after OVA sensitization. What's more, there are significantly enhanced Th2-cytokine levels, decreased IFN-γ production and increased levels of OVA-specific IgE in serum of the TGF-β1+/-mice, all of which demonstrate that the reduced expression of TGF-β1 exacerbates pathological process.

IL-10, another potent anti-inflammatory cytokine, is mainly produced by T lymphocytes, macrophages and DCs. The evidence [44] showed that IL-10 derived from CD4+T cells could inhibit pathological process through its regulation of effector responses to diseases caused by allergen and asthma. The effector responses included the inhibition of cytokine produced by Th2 cells and mast cells, eosinophil function and modulating the ratio of IgG4 and IgE. Being antigen-specific in mice sensitized with OVA, the effect of Th cells secreting IL-10 was accompanied by the increased quantities of IL-10 in BALF [45]. As far as the neutralization of IL-10 was concerned, it seriously exacerbated AHR and airway inflammation when anti-IL-10 mAb was administered [44]. IL-10 gene knockout mice were used to investigate the role of endogenous IL-10 in asthma. It turned out the reduced levels of IL-10 enhanced the airway infiltration of eosinophils and the production of IL-5, and also led to serious airway inflammation [46]. Furthermore, the expression levels of IL-4, IL-5 and IFN-γ were increased.

IFN-γ, produced by Th1 cells, inhibits many Th2-induced effector functions. Inmodels of asthma, IFN-γ reduces recruitment of lymphocytes and eosinophils, and inhibits airways hyperresponsiveness and mucus, but promotes airway neutrophils and overall lung inflammation. IFN-γ may stimulate eosinophil activation, longevity or apoptosis. Mitchell [47] used a novel transgenic mice (epi-IFN-γR mice, expresses IFN-γ receptors only on airway epithelial cells) to show the diverse airway epithelial-specific effects of IFN-γ. The results showed that IFN-γ inhibits mucus, chitinases and eosinophilia, which indicated that IFN-γ acting on the airway epithelium can inhibit Th2 cytokine effects. Furthermore, mild asthmatics inhaled IFN-γ over three weeks exhibited a reduction in airway eosinophils without evidence of an increase in proinflammatory markers. Inhaled IFN-γ could be a potential adjunctive therapy for the treatment of eosinophilic asthma.

Table 1 Application of transgenic or knockout mice to study of the cytokines

4 Conclusion

Tg animals are used in greater depth to study the pathogenesis of complicated diseases such as asthma from body, tissue and organ levels to molecule one. The models are useful for studying the pathogenesis of asthma and its pharmacological prevention while helpful to analyze the effect of a single cytokine on a single phenotype. These experiments indicate that the focused Tg models can improve understanding of the pathogenesis of asthma. The application of the transgenic mice to the cytokines in this review is summarily listed in Table 1. Furthermore, we can utilize these outcomes to guide translational studies that show the relevance of these gene expression changes in people with asthma. These mouse models can also be beneficial to the screening or the testing of the drugs to treat orprevent asthma. However, Tg animal models with asthma have their limitations. For instance, single Tg or gene-knockout mouse models may not reflect the complexities of the disease because asthma is caused by multiple genetic and environmental factors. In addition, the functions of specific proteins may not translate directly from mice to humans, on the other hand, the protein classes and cell types are still unclear that they are necessary to be investigated in detail. Therefore, further studies are needed to make Tg animals play a greater role in the investigation of the asthma pathogenesis and the development of the new therapy. In future, the studies of asthma should focus on the preparation as well as the analysis of mouse models in which multiple genes have been manipulated with Tg or gene-targeted approaches.

Fig. 1 The pathogenesis of asthma caused by the inflammatory cells and cytokines. The black arrows show the developing course and the red the process of inhibiting the development.

Acknowledgements

This study is partly supported by grants from the Education Department of Liaoning (No. L2015531) and training program of innovation for undergraduates in Shenyang Pharmaceutical University.

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* Author to whom correspondence should be addressed. Address: Professor of Pharmacology, Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, P.R. China; Tel.: +86-24-23986303; Cell phone: +86-13614053862; E-mail: 13614053862@163.com

Received: 2013-08-26 Accepted: 2015-01-20