IL-5 Transgenic Mice Research Articles

High levels of IL-7 cause dysregulation of thymocyte development

IL-7 signaling is required for thymocyte development and its loss has a severe deleterious effect on thymus function. Thymocyte-stromal cell interactions and other mechanisms tightly regulate IL-7 expression. We show that disruption of that regulation by over-expression of IL-7 inhibits T-cell development and promotes extensive B-cell lymphopoiesis in the thymus. Our data reveal that high levels of IL-7 negate Notch-1 function in thymocytes found in IL-7 transgenic mice and in co-culture with OP9-DL1 cells. While high levels of IL-7R are present on thymocytes, increased suppressor of cytokine signaling-1 expression blunts IL-7 downstream signaling, resulting in hypo-phosphorylation of proteins in the PI3K-Akt pathway. Consequently, GSK3β remains active and inhibits Notch-1 signaling as observed by decreased Hes-1 and Deltex expression in thymic progenitors. This is the first demonstration that high levels of IL-7 antagonize Notch-1 signaling and suggest that IL-7 may affect T- versus B-lineage choice in the thymus.

Interleukin-18: The Master Regulator Driving Destructive and Remodeling Processes in the Lungs of Patients with Chronic Obstructive Pulmonary Disease?

Interleukin-18: The Master Regulator Driving Destructive and Remodeling Processes in the Lungs of Patients with Chronic Obstructive Pulmonary Disease?

Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6.

Elevated levels of the inflammatory cytokine interleukin-6 (IL-6) occur in a number of CNS disorders. However, little is known about how this condition affects CNS neuronal function. Transgenic mice that express elevated levels of IL-6 in the CNS show cognitive changes, increased propensity for hippocampal seizures and reduced number of inhibitory interneurons, suggesting that elevated levels of IL-6 can cause neuroadaptive changes that alter hippocampal function. To identify these neuroadaptive changes, we measured the levels of protein expression using Western blot analysis and synaptic function using field potential recordings in hippocampus from IL-6 transgenic mice (IL-6 tg) and their non-transgenic (non-tg) littermates. Western blot analysis showed enhanced levels of the GFAP and STAT3 in the IL-6 tg hippocampus compared with the non-tg hippocampus, but no difference for several other proteins. Field potential recordings of synaptic transmission at the Schaffer collateral to CA1 synapse showed enhanced dendritic excitatory postsynaptic potentials and somatic population spikes in the CA1 region of hippocampal slices from IL-6 tg mice compared with slices from non-tg littermate controls. No differences were observed for several forms of short-term and long-term synaptic plasticity between hippocampal slices from IL-6 tg and non-tg mice. These results demonstrate that elevated levels of IL-6 can alter mechanisms involved in the excitability of hippocampal neurons and synapses, an effect consistent with recent evidence indicating that elevated production of IL-6 plays an important role in conditions associated with seizure activity and in other impairments observed in CNS disorders with a neuroinflammatory component.

TH2 cytokines increase kallikrein 7 expression and function in patients with atopic dermatitis

Here we report that Th2 cytokines increase kallikrein 7 expression and function in keratinocytes. Our finding describes a link between the Th2 environment and epidermal barrier dysfunction in atopic dermatitis.

Abstract 321: IL-6 in the tissue microenvironment plays a direct role in normal prostatic neoplastic transformation

Abstract Increased elevated levels of serum IL-6, the major inflammatory mediator, has been shown to be associated with development of progressive diseases in many types of cancers. We have previously shown that chronic exposure of immortalized benign prostate epithelial cells to paracrine IL-6 can induce tumorigenic transformation and further EMT (epithelial-mesochymal-transition)-associated metastasis in xenografts through stimulating autocrine IL-6 signaling and transactivating type I Insulin-like Growth Factor Receptor (IGF-IR). Whether IL-6, as a result of inflammation, in the tissue microenvironment can solely initiate tissue-specific de novo tumorigenesis is unclear. To address this question, we have generated a novel line of prostate-specific IL-6 transgenic mouse, in which human IL-6 (hIL-6) was expressed under the prostate-specific probasin promoter. We found that enforced expression of hIL-6 transgene in normal mouse prostate epithelium is sufficient to induce neoplastic transformation of the prostate gland, demonstrated by disappearance of base cell layer of the prostate gland and loss of E-cadherin and increased proliferation of the epithelium. Expression of the hIL-6 transgene also induced autocrine mouse IL-6 secretion in the prostate gland and sustained activation of STAT3 pathway in the epithelium and the stroma. Strikingly, IL-6 transgenic mice have increased peri-prostatic adipose tissue in which STAT3 was highly phosphorylated in comparison to the wild-type littermate. There is no significant increase in infiltration of T cells or B cells in the prostate of IL-6 transgenic animals. However, significantly increased infiltration (P<0.01) of FoxP3+ lymphocytes was seen in the stroma of the prostate in the IL-6 transgenic animals in comparison to wild-type littermates. These data suggest that IL-6 as a pro-inflammatory cytokine alone can induce tissue-specific tumorigenesis through induction of autocrine IL-6 secretion and remodeling tissue microenvironment. Our finding suggests that inflammation may play a direct role beyond being the “stimuli” or “fuel” in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 321. doi:1538-7445.AM2012-321

Stable IL-10: A New Therapeutic that Promotes Tumor Immunity

In this issue of Cancer Cell, Mumm etal. demonstrate that pegylated IL-10 increases CD8(+) Tcell numbers, IFN-γ secretion, and cytotoxicity in established tumors, enhancing antigen presentation machinery and suppressing tumor growth. This approach may enhance Tcell immune responses in cancers with reduced Tcell infiltration.

Chronic exposure to Interleukin-6 amplifies the response to Toll-like receptor ligands: implication on the pathogenesis of macrophage activation syndrome

Methods IL-6 transgenic (IL-6TG) or wild type mice were stimulated with different TLR ligands; survival, blood cells counts and biochemical parameters were analyzed. Murine splenic mononuclear cells and peritoneal macrophages from these mice were stimulated with LPS, LTA, poly I:C or CpG. Human macrophages were treated for 4 days in presence of IL-6 and then stimulated with LPS. Inflammatory cytokine expression was measured by ELISA or by RT-PCR. Activation of STAT3, ERK1/2 MAPK, p65 NFB was evaluated by Western blot or by confocal analysis.

Overexpression of chitinase 3-like 1/YKL-40 in lung-specific IL-18-transgenic mice, smokers and COPD.

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of −950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV1. Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.

The Role of TSLP in IL-13-Induced Atopic March

Although atopic dermatitis (AD) is the initial step of the “atopic march”, a progression from AD to asthma, the underlying mechanism remains unknown. Selective expression of IL-13 in the skin of mice caused an AD phenotype resembling human AD, and the disorder was associated with enhanced production of thymic stromal lymphopoietin (TSLP) in the AD skin with a systemic Th2 immunity. Here we show that IL-13 transgenic mice with AD had significantly enhanced lung inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR) when sensitized and challenged by allergen. In addition, the level of TSLP was significantly higher in acute AD than in chronic AD. Furthermore, elimination of TSLP signaling significantly diminished the allergic asthma responses, immune cell production of Th2 cytokines (IL-4, IL-13), and serum IgE. These studies indicate that IL-13 induces AD and atopic march via a TSLP dependent mechanism.

IL-13 Induces Skin Fibrosis in Atopic Dermatitis by Thymic Stromal Lymphopoietin

Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.

Antigen profiles for the quantitative assessment of eosinophils in mouse tissues by flow cytometry

Much of our current understanding of eosinophil-associated pathologies has developed from the use of mouse models. While mouse eosinophils can be readily detected by flow cytometric methods, most studies do not document the efficiency of this process compared to direct counting of stained cells. Our intent was to address this knowledge gap by identifying one or more eosinophil-specific antigen profiles that yielded flow cytometric data that was statistically consistent with direct counts. We found that anti-CD193 (CCR3) and anti-CD125 (IL-5Rα) antibodies were effective at detecting eosinophils in bone marrow of interleukin-5 transgenic mice, but these antibodies under-reported the percent positive cells. In contrast, anti-Siglec F alone or in combination with anti-CD45 can be used for the quantitative detection of eosinophils in mouse bone marrow and spleen. The antigen profile CD45 +SiglecF +CD11c − was effective at detecting eosinophils in the lung as well as bone marrow and spleen, and the results obtained correlated with direct morphometric counts under all conditions evaluated (r 2 = 0.98–0.99). To the best of our knowledge, this is the first systematic analysis presenting definitive correlations between percent eosinophils detected by cell surface markers and direct counting of stained cells in multiple tissues and at varying degrees of eosinophilia.

Development of an IL-15–autocrine CD8 T-cell leukemia in IL-15–transgenic mice requires the cis expression of IL-15Rα

AbstractIL-15 has growth-promoting effects on select lymphoid subsets, including natural killer (NK) cells, NK T cells, intraepithelial lymphocytes (IELs), CD8 T cells, and γδ-T cells. Constitutive expression of murine IL-15 in IL-15–transgenic mice was reported to cause T-NK leukemia. We investigated whether IL-15 expression is sufficient for leukemic transformation using a human IL-15–transgenic (IL-15Tg) mouse model. We noted that 100% of the mice observed over a 2-year period (n > 150) developed fatal expansions of CD8 T cells with NK markers, and determined that these cells expressed IL-15 receptor alpha (IL-15Rα). The expression of IL-15Rα on CD8 T cells appears to be required for uncontrolled aggressive lymphoproliferation, because none of the IL-15Rα−/−–IL-15Tg mice that we followed for more than 2 years developed the fatal disease despite controlled expansion of CD8 T cells. In addition, in contrast to IL-15Tg mice, in which leukemia-like CD8 T cells expressed IL-15Rα persistently, acutely activated normal CD8 T cells only transiently expressed IL-15Rα. Inhibition of DNA methylation enabled sustained IL-15Rα expression induced by activation. We present a scenario for IL-15Tg mice in which CD8 T cells that acquire constitutive persistent IL-15Rα expression are at a selective advantage and become founder cells, outgrow other lymphocytes, and lead to the establishment of a leukemia-like condition.

Eosinophils promote myocarditis and dilated cardiomyopathy by enhancing Th17 responses (101.27)

Abstract Eosinophilic myocarditis is a rare but potentially fatal form of myocarditis that is occasionally associated with systemic eosinophilia. It is not known how eosinophils localize to the myocardium or which are the mechanisms underlying their involvement in inflammatory heart diseases. Here, we assessed the role of eosinophils using a murine model of experimental autoimmune myocarditis (EAM). We have previously observed that eosinophils are recruited to the inflamed heart. Eosinophil-deficient mice (ΔdblGATA1) had decreased EAM and did not develop dilated cardiomyopathy (DCM) compared to wild-type BALB/c control mice. In contrast, IL-5 transgenic mice (IL-5Cd3) developed peripheral eosinophilia, severe EAM, increased myocardial fibrosis and accelerated DCM. Transfer of bone marrow-derived eosinophils increased EAM severity in ΔdblGATA1 mice, suggesting a direct pathogenic role for eosinophils in myocarditis. The trafficking of eosinophils to the heart was independent of IL-5 and CCR3 but partially dependent on the cytosolic phospholipase-2. Flow cytometry analysis revealed that the frequency of IL-17A-secreting T cells tracked with the number of eosinophils; it was significantly increased in IL-5Cd3 and decreased in ΔdblGATA1 mice. This study provides the first direct evidence for a pathogenic role in eosinophils in myocarditis and DCM. Additionally, our findings support the hypothesis that eosinophils may promote Th17 responses during myocarditis.

NKp46 identifies an NKT cell subset susceptible to leukemic transformation in mouse and human

IL-15 may have a role in the development of T cell large granular lymphocyte (T-LGL) or NKT leukemias. However, the mechanisms of action and the identity of the cell subset that undergoes leukemic transformation remain elusive. Here we show that in both mice and humans, NKp46 expression marks a minute population of WT NKT cells with higher activity and potency to become leukemic. Virtually 100% of T-LGL leukemias in IL-15 transgenic mice expressed NKp46, as did a majority of human T-LGL leukemias. The minute NKp46+ NKT population, but not the NKp46⁻ NKT population, was selectively expanded by overexpression of endogenous IL-15. Importantly, IL-15 transgenic NKp46⁻ NKT cells did not become NKp46+ in vivo, suggesting that NKp46+ T-LGL leukemia cells were the malignant counterpart of the minute WT NKp46+ NKT population. Mechanistically, NKp46+ NKT cells possessed higher responsiveness to IL-15 in vitro and in vivo compared with that of their NKp46⁻ NKT counterparts. Furthermore, interruption of IL-15 signaling using a neutralizing antibody could prevent LGL leukemia in IL-15 transgenic mice. Collectively, our data demonstrate that NKp46 identifies a functionally distinct NKT subset in mice and humans that appears to be directly susceptible to leukemic transformation when IL-15 is overexpressed. Thus, IL-15 signaling and NKp46 may be useful targets in the treatment of patients with T-LGL or NKT leukemia.

A novel ELISA for eosinophil peroxidase provides a sensitive high throughput assay for eosinophil degranulation in either mouse or human biological samples (65.29)

Abstract Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.

The Influence of Excessive IL-6 Production In Vivo on the Development and Function of Foxp3+ Regulatory T Cells

IL-6 is a proinflammatory cytokine and its overproduction is implicated in a variety of inflammatory disorders. Recent in vitro analyses suggest that IL-6 is a key cytokine that determines the balance between Foxp3(+) regulatory T cells (Tregs) and Th17 cells. However, it remains unclear whether excessive IL-6 production in vivo alters the development and function of Foxp3(+) Tregs. In this study, we analyzed IL-6 transgenic (Tg) mice in which serum IL-6 levels are constitutively elevated. Interestingly, in IL-6 Tg mice, whereas peripheral lymphoid organs were enlarged, and T cells exhibited activated phenotype, Tregs were not reduced but rather increased compared with wild-type mice. In addition, Tregs from Tg mice normally suppressed proliferation of naive T cells in vitro. Furthermore, Tregs cotransferred with naive CD4 T cells into SCID-IL-6 Tg mice inhibited colitis as successfully as those transferred into control SCID mice. These results indicate that overproduction of IL-6 does not inhibit development or function of Foxp3(+) Tregs in vivo. However, when naive CD4 T cells alone were transferred, Foxp3(+) Tregs retrieved from SCID-IL-6 Tg mice were reduced compared with SCID mice. Moreover, the Helios(-) subpopulation of Foxp3(+) Tregs, recently defined as extrathymic Tregs, was significantly reduced in IL-6 Tg mice compared with wild-type mice. Collectively, these results suggest that IL-6 overproduced in vivo inhibits inducible Treg generation from naive T cells, but does not affect the development and function of natural Tregs.

FTY720 Regulates Bone Marrow Egress of Eosinophils and Modulates Late-Phase Skin Reaction in Mice

Eosinophilia in the blood and skin is frequently observed in patients with certain inflammatory skin diseases, such as atopic dermatitis. However, the mechanism underlying eosinophil circulation and the role of eosinophils in cutaneous immune responses remain unclear. In repeated hapten application-induced cutaneous responses in BALB/c mice, the administration of FTY720 before the last challenge decreased the number of skin-infiltrating eosinophils and reduced the late-phase reaction. A similar reduction of the late-phase reaction was observed by a sphingosine-1-phosphate G protein-coupled receptor (S1P1)-selective agonist, SEW2871. We monitored numerous alterations of eosinophils in the blood, spleen, bone marrow, and lymph nodes of interleukin-5 transgenic mice, used as an eosinophilia model, following FTY720 administration. The number of circulating eosinophils was significantly decreased after treatment with FTY720, and eosinophils accumulated in the bone marrow. In addition, eosinophils expressed S1P1, S1P3, and S1P4 mRNAs, and their chemotactic response to S1P was abolished by FTY720 as well as by SEW2871. These findings suggest that FTY720 affects the number of eosinophils in both the blood and skin by inhibiting the egress of eosinophils from the bone marrow and thus downmodulating the late-phase reaction.

IL-7–Dependent B Lymphocytes Are Essential for the Anti-polysaccharide Response and Protective Immunity toStreptococcus pneumoniae

Young children are impaired in their response to T cell-independent (TI) Ags, such as pneumococcal polysaccharide (PPS). B lymphopoeisis early in life is IL-7 independent, whereas in adults it is IL-7 dependent. Therefore, we hypothesized that IL-7-driven B lymphopoiesis plays a critical role in promoting Ab responses to TI Ags. Young but not adult mice are impaired in responses to PPS vaccination and to 4-hydroxy-3-nitrophenyl-acetyl-Ficoll, a widely studied model TI Ag, and B1b cells generate Ab responses to these Ags. In this paper, we show that, despite having B1b, B1a, and MZ B cells-all of which are involved in TI responses-young wild-type or adult mice deficient either in IL-7 or in IL-7Ralpha are severely impaired in anti-PPS responses and do not survive Streptococcus pneumoniae challenge, indicating IL-7-dependent B cells are required for TI immunity. Consistent with this, PPS immunization induced a robust TI response in young IL-7 transgenic mice that was comparable to adult wild-type responses. Moreover, immunized young or adult IL-7 transgenic mice were completely resistant to S. pneumoniae challenge. Our data indicate that activating the IL-7 signaling pathway could restore impaired TI responses in the young.

IL-13 Induces Esophageal Remodeling and Gene Expression by an Eosinophil-Independent, IL-13Rα2–Inhibited Pathway

Eosinophilic esophagitis (EE) is an emerging disease associated with both food and respiratory allergy characterized by extensive esophageal tissue remodeling and abnormal esophageal gene expression, including increased IL-13. We investigated the ability of increased airway IL-13 to induce EE-like changes. Mice with pulmonary (but not esophageal) overexpression of IL-13 evidenced esophageal IL-13 accumulation and developed prominent esophageal remodeling with epithelial hyperplasia, angiogenesis, collagen deposition, and increased circumference. IL-13 induced notable changes in esophageal transcripts that overlapped with the human EE esophageal transcriptome. IL-13-induced esophageal eosinophilia was dependent on eotaxin-1 (but not eotaxin-2). However, remodeling occurred independent of eosinophils as demonstrated by eosinophil lineage-deficient, IL-13 transgenic mice. IL-13-induced remodeling was significantly enhanced by IL-13Ralpha2 deletion, indicating an inhibitory effect of IL-13Ralpha2. In the murine system, there was partial overlap between IL-13-induced genes in the lung and esophagus, yet the transcriptomes were divergent at the tissue level. In human esophagus, IL-13 levels correlated with the magnitude of the EE transcriptome. In conclusion, inducible airway expression of IL-13 results in a pattern of esophageal gene expression and extensive tissue remodeling that resembles human EE. Notably, we identified a pathway that induces EE-like changes and is IL-13-driven, eosinophil-independent, and suppressed by IL-13Ralpha2.

FC-OPG prevents IL-6 dependent bone loss and impaired skeletal growth in IL-6 transgenic mice. implications for therapy in juvenile chronic inflammatory diseases

FC-OPG prevents IL-6 dependent bone loss and impaired skeletal growth in IL-6 transgenic mice. implications for therapy in juvenile chronic inflammatory diseases