Pinyi Lu

Systems modeling of molecular mechanisms controlling cytokine-driven CD4+ T cell differentiation and phenotype plasticity.

Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa.

Abscisic Acid Regulates Inflammation via Ligand-binding Domain-independent Activation of Peroxisome Proliferator-activated Receptor γ

Abscisic acid (ABA) has shown efficacy in the treatment of diabetes and inflammation; however, its molecular targets and the mechanisms of action underlying its immunomodulatory effects remain unclear. This study investigates the role of peroxisome proliferator-activated receptor γ (PPAR γ) and lanthionine synthetase C-like 2 (LANCL2) as molecular targets for ABA. We demonstrate that ABA increases PPAR γ reporter activity in RAW 264.7 macrophages and increases ppar γ expression in vivo, although it does not bind to the ligand-binding domain of PPAR γ. LANCL2 knockdown studies provide evidence that ABA-mediated activation of macrophage PPAR γ is dependent on lancl2 expression. Consistent with the association of LANCL2 with G proteins, we provide evidence that ABA increases cAMP accumulation in immune cells. ABA suppresses LPS-induced prostaglandin E2 and MCP-1 production via a PPAR γ-dependent mechanism possibly involving activation of PPAR γ and suppression of NF-κB and nuclear factor of activated T cells. LPS challenge studies in PPAR γ-expressing and immune cell-specific PPAR γ null mice demonstrate that ABA down-regulates toll-like receptor 4 expression in macrophages and T cells in vivo through a PPAR γ-dependent mechanism. Global transcriptomic profiling and confirmatory quantitative RT-PCR suggest novel candidate targets and demonstrate that ABA treatment mitigates the effect of LPS on the expression of genes involved in inflammation, metabolism, and cell signaling, in part, through PPAR γ. In conclusion, ABA decreases LPS-mediated inflammation and regulates innate immune responses through a bifurcating pathway involving LANCL2 and an alternative, ligand-binding domain-independent mechanism of PPAR γ activation.

Preventive and Prophylactic Mechanisms of Action of Pomegranate Bioactive Constituents

Pomegranate fruit presents strong anti-inflammatory, antioxidant, antiobesity, and antitumoral properties, thus leading to an increased popularity as a functional food and nutraceutical source since ancient times. It can be divided into three parts: seeds, peel, and juice, all of which seem to have medicinal benefits. Several studies investigate its bioactive components as a means to associate them with a specific beneficial effect and develop future products and therapeutic applications. Many beneficial effects are related to the presence of ellagic acid, ellagitannins (including punicalagins), punicic acid and other fatty acids, flavonoids, anthocyanidins, anthocyanins, estrogenic flavonols, and flavones, which seem to be its most therapeutically beneficial components. However, the synergistic action of the pomegranate constituents appears to be superior when compared to individual constituents. Promising results have been obtained for the treatment of certain diseases including obesity, insulin resistance, intestinal inflammation, and cancer. Although moderate consumption of pomegranate does not result in adverse effects, future studies are needed to assess safety and potential interactions with drugs that may alter the bioavailability of bioactive constituents of pomegranate as well as drugs. The aim of this review is to summarize the health effects and mechanisms of action of pomegranate extracts in chronic inflammatory diseases.

Molecular modeling of lanthionine synthetase component C-like protein 2: a potential target for the discovery of novel type 2 diabetes prophylactics and therapeutics

The rates of type 2 diabetes (T2D) are rising to epidemic proportions in the US and worldwide. While current T2D medications are efficacious, significant side effects have limited their use and availability. Our laboratory has discovered that abscisic acid (ABA) exerts anti-diabetic effects, in part, by activating peroxisome proliferator-activated receptor γ (PPAR γ). However, since ABA does not bind to the ligand-binding domain (LBD) of PPAR γ, the mechanism of activation of PPAR γ by ABA remains unknown. Lanthionine synthetase component C-like protein 2 (LANCL2) was predicted to be a novel target for the binding and signaling of ABA in human granulocytes and rat insulinoma cells. The goal of this study was to determine whether LANCL2 is a molecular target of ABA and other PPAR γ agonists. To this end we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of LANCL1 as a template. Our molecular docking studies predicted that ABA and other PPAR γ agonists (e.g., rosiglitazone and pioglitazone) share a binding site on the surface of LANCL2. The identification of a binding site for PPAR γ agonists will facilitate the high-throughput virtual screening of large compound libraries and may shed new light on alternative mechanisms of PPAR γ activation.

Computational Modeling-Based Discovery of Novel Classes of Anti-Inflammatory Drugs That Target Lanthionine Synthetase C-Like Protein 2

Background Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects. Methodology/Principal Findings The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses. Conclusions/Significance LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.

Dietary α-Eleostearic Acid Ameliorates Experimental Inflammatory Bowel Disease in Mice by Activating Peroxisome Proliferator-Activated Receptor-γ

Background Treatments for inflammatory bowel disease (IBD) are modestly effective and associated with side effects from prolonged use. As there is no known cure for IBD, alternative therapeutic options are needed. Peroxisome proliferator-activated receptor-gamma (PPARγ) has been identified as a potential target for novel therapeutics against IBD. For this project, compounds were screened to identify naturally occurring PPARγ agonists as a means to identify novel anti-inflammatory therapeutics for experimental assessment of efficacy. Methodology/Principal Findings Here we provide complementary computational and experimental methods to efficiently screen for PPARγ agonists and demonstrate amelioration of experimental IBD in mice, respectively. Computational docking as part of virtual screening (VS) was used to test binding between a total of eighty-one compounds and PPARγ. The test compounds included known agonists, known inactive compounds, derivatives and stereoisomers of known agonists with unknown activity, and conjugated trienes. The compound identified through VS as possessing the most favorable docked pose was used as the test compound for experimental work. With our combined methods, we have identified α-eleostearic acid (ESA) as a natural PPARγ agonist. Results of ligand-binding assays complemented the screening prediction. In addition, ESA decreased macrophage infiltration and significantly impeded the progression of IBD-related phenotypes through both PPARγ-dependent and –independent mechanisms in mice with experimental IBD. Conclusions/Significance This study serves as the first significant step toward a large-scale VS protocol for natural PPARγ agonist screening that includes a massively diverse ligand library and structures that represent multiple known target pharmacophores.

Lanthionine Synthetase Component C-Like Protein 2: A New Drug Target for Inflammatory Diseases and Diabetes

Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the LANCL protein family, which is broadly expressed throughout the body. LANCL2 is the molecular target of abscisic acid (ABA), a compound with insulin-sensitizing and immune modulatory actions. LANCL2 is required for membrane binding and signaling of ABA in immune cells. Direct binding of ABA to LANCL2 was predicted in silico using molecular modeling approaches and validated experimentally using ligand-binding assays and kinetic surface plasmon resonance studies. The therapeutic potential of the LANCL2 pathway ranges from increasing cellular sensitivity to anticancer drugs, insulin-sensitizing effects and modulating immune and inflammatory responses in the context of immune-mediated and infectious diseases. A case for LANCL2-based drug discovery and development is also illustrated by the anti-inflammatory activity of novel LANCL2 ligands such as NSC61610 against inflammatory bowel disease and influenza-driven inflammation in mice. This review discusses the value of LANCL2 as a novel therapeutic target for the discovery and development of new classes of orally active drugs against chronic metabolic, immune-mediated and infectious diseases.

Modeling the Role of Lanthionine Synthetase C-Like 2 (LANCL2) in the Modulation of Immune Responses to Helicobacter pylori Infection

Immune responses to Helicobacter pylori are orchestrated through complex balances of host-bacterial interactions, including inflammatory and regulatory immune responses across scales that can lead to the development of the gastric disease or the promotion of beneficial systemic effects. While inflammation in response to the bacterium has been reasonably characterized, the regulatory pathways that contribute to preventing inflammatory events during H. pylori infection are incompletely understood. To aid in this effort, we have generated a computational model incorporating recent developments in the understanding of H. pylori-host interactions. Sensitivity analysis of this model reveals that a regulatory macrophage population is critical in maintaining high H. pylori colonization without the generation of an inflammatory response. To address how this myeloid cell subset arises, we developed a second model describing an intracellular signaling network for the differentiation of macrophages. Modeling studies predicted that LANCL2 is a central regulator of inflammatory and effector pathways and its activation promotes regulatory responses characterized by IL-10 production while suppressing effector responses. The predicted impairment of regulatory macrophage differentiation by the loss of LANCL2 was simulated based on multiscale linkages between the tissue-level gastric mucosa and the intracellular models. The simulated deletion of LANCL2 resulted in a greater clearance of H. pylori, but also greater IFNγ responses and damage to the epithelium. The model predictions were validated within a mouse model of H. pylori colonization in wild-type (WT), LANCL2 whole body KO and myeloid-specific LANCL2-/- (LANCL2Myeloid) mice, which displayed similar decreases in H. pylori burden, CX3CR1+ IL-10-producing macrophages, and type 1 regulatory (Tr1) T cells. This study shows the importance of LANCL2 in the induction of regulatory responses in macrophages and T cells during H. pylori infection.

Lanthionine Synthetase C-Like 2 Modulates Immune Responses to Influenza Virus Infection

Broad-based, host-targeted therapeutics have the potential to ameliorate viral infections without inducing antiviral resistance. We identified lanthionine synthetase C-like 2 (LANCL2) as a new therapeutic target for immunoinflammatory diseases. To examine the therapeutic efficacy of oral NSC61610 administration on influenza, we infected C57BL/6 mice with influenza A H1N1pdm virus and evaluated influenza-related mortality, lung inflammatory profiles, and pulmonary histopathology. Oral treatment with NSC61610 ameliorates influenza virus infection by down-modulating pulmonary inflammation through the downregulation of TNF-α and MCP-1 and reduction in the infiltration of neutrophils. NSC61610 treatment increases IL10-producing CD8+ T cells and macrophages in the lungs during the resolution phase of disease. The loss of LANCL2 or neutralization of IL-10 in mice infected with influenza virus abrogates the ability of NSC61610 to accelerate recovery and induce IL-10-mediated regulatory responses. These studies validate that oral treatment with NSC61610 ameliorates morbidity and mortality and accelerates recovery during influenza virus infection through a mechanism mediated by activation of LANCL2 and subsequent induction of IL-10 responses by CD8+ T cells and macrophages in the lungs.

Latent-Based Imputation of Laboratory Measures from Electronic Health Records: Case for Complex Diseases

Imputation is a key step in Electronic Health Records-mining as it can significantly affect the conclusions derived from the downstream analysis. There are three main categories that explain the missingness in clinical settings–incompleteness, inconsistency, and inaccuracy–and these can capture a variety of situations: the patient did not seek treatment, the health care provider did not enter the information, etc. We used EHR data from patients diagnosed with Inflammatory Bowel Disease from Geisinger Health System to design a novel imputation that focuses on a complex phenotype. Our approach is based on latent-based analysis integrated with clustering to group patients based on their comorbidities before imputation. IBD is a chronic illness of unclear etiology and without a complete cure. We have taken advantage of the complexity of IBD to pre-process the EHR data of 10,498 IBD patients and show that imputation can be improved using shared latent comorbidities. The R code and sample simulated input data will be available at a future time.