Mythili Dileepan

MicroRNA Regulation of Airway Inflammation and Airway Smooth Muscle Function: Relevance to Asthma.

Preclinical Research

MicroRNA-708 regulates CD38 expression through signaling pathways JNK MAP kinase and PTEN/AKT in human airway smooth muscle cells

BackgroundThe cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38−/− mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3’ Untranslated Region (3’UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells.MethodsGrowth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3’UTR to alter gene expression.ResultsUsing target prediction algorithms, we identified several miRNAs with potential CD38 3’UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3’UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling.ConclusionsIn human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3’UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.

MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells

Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

Altered CD38/Cyclic ADP-Ribose Signaling Contributes to the Asthmatic Phenotype

CD38 is a transmembrane glycoprotein expressed in airway smooth muscle cells. The enzymatic activity of CD38 generates cyclic ADP-ribose from β-NAD. Cyclic ADP-ribose mobilizes intracellular calcium during activation of airway smooth muscle cells by G-protein-coupled receptors through activation of ryanodine receptor channels in the sarcoplasmic reticulum. Inflammatory cytokines that are implicated in asthma upregulate CD38 expression and increase the calcium responses to contractile agonists in airway smooth muscle cells. The augmented intracellular calcium responses following cytokine exposure of airway smooth muscle cells are inhibited by an antagonist of cyclic ADP-ribose. Airway smooth muscle cells from CD38 knockout mice exhibit attenuated intracellular calcium responses to agonists, and these mice have reduced airway response to inhaled methacholine. CD38 also contributes to airway hyperresponsiveness as shown in mouse models of allergen or cytokine-induced inflammatory airway disease. In airway smooth muscle cells obtained from asthmatics, the cytokine-induced CD38 expression is significantly enhanced compared to expression in cells from nonasthmatics. This differential induction of CD38 expression in asthmatic airway smooth muscle cells stems from increased activation of MAP kinases and transcription through NF-κB, and altered post-transcriptional regulation through microRNAs. We propose that increased capacity for CD38 signaling in airway smooth muscle in asthma contributes to airway hyperresponsiveness.

A Novel Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and Cellular Immunity after a Booster Dose

ABSTRACT Pichinde virus (PICV) is a bisegmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV (strain P18) with a trisegmented RNA genome (rP18tri), which encodes all four PICV gene products and as many as two foreign genes. We have engineered the vector to express the green fluorescent protein (GFP) reporter gene (abbreviated as G in virus designations) and either the hemagglutination (HA [H]) or the nucleoprotein (NP [P]) gene of the influenza A/PR8 virus. The trisegmented viruses rP18tri-G/H and rP18tri-G/P showed slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza virus challenge even 120 days after immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a prime-and-boost vaccination strategy. IMPORTANCE We have developed a novel Pichinde virus (PICV)-based live viral vector, rP18tri, that packages three RNA segments and encodes as many as two foreign genes. Using the influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and can lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that distinguishes this from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.

Inhibition of soluble epoxide hydrolase attenuates eosinophil recruitment and food allergen‐induced gastrointestinal inflammation

Prevalence of food allergies in the United States is on the rise. Eosinophils are recruited to the intestinal mucosa in substantial numbers in food allergen‐driven gastrointestinal (GI) inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro‐inflammatory role during inflammation by metabolizing anti‐inflammatory epoxyeicosatrienoic acids (EETs) to pro‐inflammatory diols. We investigated the role of sEH in a murine model of food allergy and evaluated the potential therapeutic effect of a highly selective sEH inhibitor (trans‐4‐{4‐[3‐(4‐trifluoromethoxyphenyl)‐ureido]‐cyclohexyloxy}‐benzoic acid [t‐TUCB]). Oral exposure of mice on a soy‐free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen‐specific IgE levels, and caused significant weight loss. Administration of t‐TUCB to SPI‐challenged mice inhibited IgE levels and prevented SPI‐induced weight loss. Additionally, SPI‐induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin 1 levels, mucus hypersecretion, and decreased epithelial junction protein expression. In t‐TUCB‐treated mice, eosinophilia, mast cell recruitment, and mucus secretion were significantly lower than in untreated mice and SPI‐induced loss of junction protein expression was prevented to variable levels. sEH expression in eosinophils was induced by inflammatory mediators TNF‐α and eotaxin‐1. Treatment of eosinophils with t‐TUCB significantly inhibited eosinophil migration, an effect that was mirrored by treatment with 11,12‐EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin‐1‐induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses and GI inflammation including eosinophilia and that inhibition of sEH can attenuate these responses.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation

Background: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. Objective: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. Methods: Airway inflammation, including junction protein expression, was evaluated in allergen‐challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. Results: Administration of knob to allergen‐challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL‐5 levels) and prevented allergen‐induced loss of airway epithelial occludin and E‐cadherin expression. Additionally, knob decreased expression of TH2‐promoting inflammatory mediators, specifically IL‐33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c‐Jun N‐terminal kinase, increased expression of occludin and E‐cadherin, and enhanced epithelial barrier integrity. Conclusion: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen‐induced airway inflammatory response, including asthma.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced β2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

CD38-Cyclic ADP-Ribose-Mediated Calcium Signaling in Airway Myocytes

Nicotinamide adenine dinucleotide (NAD) metabolites, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP) have been identified as calcium-releasing second messengers. In smooth muscle including that of airways, cADPR plays a vital role in the dynamic regulation of intracellular calcium and contraction. CD38, a 45 kDa bifunctional transmembrane protein, possesses enzymatic activities (ADP-ribosyl cyclase and cADPR hydrolase) necessary for the synthesis and degradation of cADPR. Together, CD38 and cADPR form a signaling cascade in agonist-induced calcium elevation in airway smooth muscle (ASM) cells similar to well-established phospholipase C and inositol trisphosphate (PLC/IP3) pathway. CD38/cADPR is considered an endogenous activator of calcium release from the sarcoplasmic reticulum via ryanodine receptor channels. Most importantly, findings from ex vivo and in vivo studies have established the contribution of CD38/cADPR-mediated calcium release to the regulation of contractile responsiveness of airways and respiratory function. CD38 expression is regulated by inflammatory cytokines, microRNAs, and exogenous drugs such as corticosteroids. Changes in CD38 expression and cADPR production have significant consequences in ASM functions and also contribute to hyperresponsiveness seen during airway inflammatory conditions such as asthma. This chapter describes numerous studies that have established signaling, functional, and pathophysiological roles of CD38/cADPR in ASM.