Thomas X. Lu

MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression

Allergic airway inflammation is characterized by marked in situ changes in gene and protein expression, yet the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in this process has not yet been reported. Using a highly sensitive microarray-based approach, we identified 21 miRNAs with differential expression between doxycycline-induced lung-specific IL-13 transgenic mice (with allergic airway inflammation) and control mice. In particular, we observed overexpression of miR-21 and underexpression of miR-1 in the induced IL-13 transgenic mice compared with control mice. These findings were validated in two independent models of allergen-induced allergic airway inflammation and in IL-4 lung transgenic mice. Although IL-13-induced miR-21 expression was IL-13Rα1 dependent, allergen-induced miR-21 expression was mediated mainly independent of IL-13Rα1 and STAT6. Notably, predictive algorithms identified potential direct miR-21 targets among IL-13-regulated lung transcripts, such as IL-12p35 mRNA, which was decreased in IL-13 transgenic mice. Introduction of pre-miR-21 dose dependently inhibited cellular expression of a reporter vector harboring the 3′-untranslated region of IL-12p35. Moreover, mutating miR-21 binding sites in IL-12p35 3′-untranslated region abrogated miR-21-mediated repression. In summary, we have identified a miRNA signature in allergic airway inflammation, which includes miR-21 that modulates IL-12, a molecule germane to Th cell polarization.

MicroRNA-21 limits in vivo immune response-mediated activation of the IL-12/IFN-gamma pathway, Th1 polarization, and the severity of delayed-type hypersensitivity.

An altered balance between Th1 and Th2 cytokines is responsible for a variety of immunoinflammatory disorders such as asthma, yet the role of posttranscriptional mechanisms, such as those mediated by microRNAs (miRs), in adjusting the relative magnitude and balance of Th cytokine expression have been largely unexplored. In this study, we show that miR-21 has a central role in setting a balance between Th1 and Th2 responses to Ags. Targeted ablation of miR-21 in mice led to reduced lung eosinophilia after allergen challenge, with a broadly reprogrammed immunoactivation transcriptome and significantly increased levels of the Th1 cytokine IFN-γ. Biological network-based transcriptome analysis of OVA-challenged miR-21−/− mice identified an unexpected prominent dysregulation of IL-12/IFN-γ pathways as the most significantly affected in the lungs, with a key role for miR-21 in IFN-γ signaling and T cell polarization, consistent with a functional miR-21 binding site in IL-12p35. In support of these hypotheses, miR-21 deficiency led dendritic cells to produce more IL-12 after LPS stimulation and OVA-challenged CD4+ T lymphocytes to produce increased IFN-γ and decreased IL-4. Further, loss of miR-21 significantly enhanced the Th1-associated delayed-type hypersensitivity cutaneous responses. Thus, our results define miR-21 as a major regulator of Th1 versus Th2 responses, defining a new mechanism for regulating polarized immunoinflammatory responses.

Diagnostic, functional, and therapeutic roles of microRNA in allergic diseases

Allergic inflammation is accompanied by the coordinated expression of a myriad of genes and proteins that initiate, sustain, and propagate immune responses and tissue remodeling. MicroRNAs (miRNAs) are a class of short single-stranded RNA molecules that posttranscriptionally silence gene expression and have been shown to fine-tune gene transcriptional networks because single miRNAs can target hundreds of genes. Considerable attention has been focused on the key role of miRNAs in regulating homeostatic immune architecture and acquired immunity. Recent studies have identified miRNA profiles in multiple allergic inflammatory diseases, including asthma, eosinophilic esophagitis, allergic rhinitis, and atopic dermatitis. Specific miRNAs have been found to have critical roles in regulating key pathogenic mechanisms in allergic inflammation, including polarization of adaptive immune responses and activation of T cells (eg, miR-21 and miR-146), regulation of eosinophil development (eg, miR-21 and miR-223), and modulation of IL-13-driven epithelial responses (eg, miR-375). This review discusses recent advances in our understanding of the expression and function of miRNAs in patients with allergic inflammation, their role as disease biomarkers, and perspectives for future investigation and clinical utility.

MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.

BACKGROUND The role of microRNAs (miRNAs), a key class of regulators of mRNA expression and translation, in patients with eosinophilic esophagitis (EoE) has not been explored. OBJECTIVE We aimed to identify miRNAs dysregulated in patients with EoE and assess the potential of these miRNAs as disease biomarkers. METHODS Esophageal miRNA expression was profiled in patients with active EoE and those with glucocorticoid-induced disease remission. Expression profiles were compared with those of healthy control subjects and patients with chronic (noneosinophilic) esophagitis. Expression levels of the top differentially expressed miRNAs from the plasma of patients with active EoE and patients with EoE remission were compared with those of healthy control subjects. RESULTS EoE was associated with 32 differentially regulated miRNAs and was distinguished from noneosinophilic forms of esophagitis. The expression levels of the most upregulated miRNAs (miR-21 and miR-223) and the most downregulated miRNA (miR-375) strongly correlated with esophageal eosinophil levels. Bioinformatic analysis predicted interplay of miR-21 and miR-223 with key roles in the polarization of adaptive immunity and regulation of eosinophilia, and indeed, these miRNAs correlated with key elements of the EoE transcriptome. The differentially expressed miRNAs were largely reversible in patients who responded to glucocorticoid treatment. EoE remission induced a single miRNA (miR-675) likely to be involved in DNA methylation. Plasma analysis of the most upregulated esophageal miRNAs identified miR-146a, miR-146b, and miR-223 as the most differentially expressed miRNAs in the plasma. CONCLUSIONS We have identified a marked dysregulated expression of a select group of miRNAs in patients with EoE and defined their reversibility with glucocorticoid treatment and their potential value as invasive and noninvasive biomarkers.

MiR-375 is downregulated in epithelial cells after IL-13 stimulation and regulates an IL-13-induced epithelial transcriptome.

Interleukin 13 (IL-13)-induced epithelial gene and protein expression changes are central to the pathogenesis of multiple allergic diseases. Herein, using human esophageal squamous and bronchial columnar epithelial cells, we identified microRNAs (miRNAs) that were differentially regulated after IL-13 stimulation. Among the IL-13-regulated miRNAs, miR-375 showed a conserved pattern of downregulation. Furthermore, miR-375 was downregulated in the lung of IL-13 lung transgenic mice. We subsequently analyzed miR-375 levels in a human disease characterized by IL-13 overproduction—the allergic disorder eosinophilic esophagitis (EE)—and observed downregulation of miR-375 in EE patient samples compared with control patients. MiR-375 expression levels reflected disease activity, normalized with remission, and inversely correlated with the degree of allergic inflammation. Using a lentiviral strategy and whole-transcriptome analysis in epithelial cells, miR-375 overexpression was sufficient to markedly modify IL-13-associated immunoinflammatory pathways in epithelial cells in vitro, further substantiating interactions between miR-375 and IL-13. Taken together, our results support a key role of miRNAs, particularly miR-375, in regulating and fine-tuning IL-13-mediated responses.

miR-223 Deficiency Increases Eosinophil Progenitor Proliferation

Recently, microRNAs have been shown to be involved in hematopoietic cell development, but their role in eosinophilopoiesis has not yet been described. In this article, we show that miR-223 is upregulated during eosinophil differentiation in an ex vivo bone marrow–derived eosinophil culture system. Targeted ablation of miR-223 leads to an increased proliferation of eosinophil progenitors. We found upregulation of a miR-223 target gene, IGF1R, in the eosinophil progenitor cultures derived from miR-223−/− mice compared with miR-223+/+ littermate controls. The increased proliferation of miR-223−/− eosinophil progenitors was reversed by treatment with an IGF1R inhibitor (picropodophyllin). Whole-genome microarray analysis of differentially regulated genes between miR-223+/+ and miR-223−/− eosinophil progenitor cultures identified a specific enrichment in genes that regulate hematologic cell development. Indeed, miR-223−/− eosinophil progenitors had a delay in differentiation. Our results demonstrate that microRNAs regulate the development of eosinophils by influencing eosinophil progenitor growth and differentiation and identify a contributory role for miR-223 in this process.

Targeted Ablation of miR-21 Decreases Murine Eosinophil Progenitor Cell Growth

MiR-21 is one of the most up-regulated miRNAs in multiple allergic diseases associated with eosinophilia and has been shown to positively correlate with eosinophil levels. Herein, we show that miR-21 is up-regulated during IL-5-driven eosinophil differentiation from progenitor cells in vitro. Targeted ablation of miR-21 leads to reduced eosinophil progenitor cell growth. Furthermore, miR-21−/− eosinophil progenitor cells have increased apoptosis as indicated by increased levels of annexin V positivity compared to miR-21+/+ eosinophil progenitor cells. Indeed, miR-21−/− mice have reduced blood eosinophil levels in vivo and reduced eosinophil colony forming unit capacity in the bone marrow. Using gene expression microarray analysis, we identified dysregulation of genes involved in cell proliferation (e,g, Ms4a3, Grb7), cell cycle and immune response as the most significant pathways affected by miR-21 in eosinophil progenitors. These results demonstrate that miR-21 can regulate the development of eosinophils by influencing eosinophil progenitor cell growth. Our findings have identified one of the first miRNAs with a role in regulating eosinophil development.

Epigenetic Regulation of the IL-13-induced Human Eotaxin-3 Gene by CREB-binding Protein-mediated Histone 3 Acetylation

The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetyltransferase, induced base-line and IL-13-induced eotaxin-3 promoter activity. Additionally, IL-13 treatment promoted global histone 3 acetylation as well as the formation of a complex containing CBP and STAT6 and the subsequent acetylation of histone 3 at the eotaxin-3 promoter. CBP gene silencing decreased IL-13-induced transcription of eotaxin-3. Conversely, inhibition of histone deacetylation increased IL-13-induced eotaxin-3 production. Clinical studies demonstrated markedly increased global acetylation of histone 3 in the inflamed tissue of patients with allergic inflammation. Collectively, these results identify an epigenetic mechanism involving CBP and chromatin remodeling in regulating IL-13-induced chemokine transcription.

515 microRNA-375-KLF5 Regulation of Intestinal Epithelial CFTR Function

Background: Matrix Metalloproteinase (MMP)-induced extracellular matrix remodeling modulates intestinal inflammation. MMP-12 is a human macrophage elastase capable of degrading basement membrane (BM). Defective intestinal barrier leading to increased intestinal permeability is an important pathogenic factor for intestinal inflammation. The role of MMP-12 in intestinal barrier function and intestinal inflammation remains unclear. We hypothesize that MMP-12 induces degradation of BM and helps macrophage transmigration thereby compromising intestinal barrier and augmenting intestinal inflammation. Aim: The aim of this study was to investigate the role of MMP-12 in intestinal epithelial tight junction (TJ) permeability and macrophage transmigration in experimental dextran sodium sulfate (DSS) colitis and in vitro Caco-2 cell model. Methods: Nine weeks-old wild type (WT) and MMP-12-/mice were administered 3% DSS in drinking water for 7 days. An in vivo colonic recycling perfusion and in vitro epithelial cell model was used to study epithelial permeability. Results: DSS treatment resulted in a marked increase in MMP-12 protein expression in WT colon. DSS administration significantly increased the colonic permeability in WT but not MMP-12-/mice (p<0.01). The loss of body weight, disease activity index, and histological lesion score of colitis was significantly attenuated in MMP-12-/DSS group compared to WT DSS group (p<0.01). In immunohistochemical study, the BM laminin was significantly lost in WT DSS colon but not in MMP-12-/DSS colon. The epithelial infiltration of macrophages in DSS colitis, as assessed by macrophage marker CD68 staining, was found to be significantly lower in MMP-12-/mice than WT mice. To further investigate the role of MMP-12 in intestinal TJ permeability, human intestinal epithelial Caco-2 cells were co-cultured with phorbol myristate acetate activated, MMP-12 secreting human macrophage U937 cells. The co-culture resulted into progressive and significant decrease in Caco-2 transepithelial resistance (TER) and concomitant increase in paracellular inulin flux, indicating loss of Caco-2 TJ barrier. Furthermore, siRNA-induced knock down of MMP-12 expression in U937 macrophage cells attenuated loss of Caco-2 TER and increase in inulin flux after U937 co-culture. Also, siRNA-induced knock down of MMP-12 significantly prevented U937 macrophage transmigration across the Caco-2 cells. Conclusion:The clinical severity, colonic permeability, and macrophage infiltration in colitis was attenuated in MMP-12-/mice. Macrophage derived MMP-12 increased intestinal epithelial permeability and enables macrophage transmigration in a cell culture model. These data suggest that MMP-12-induced macrophage transmigration and loss of intestinal epithelial TJ barrier contributes to the development of colitis.