Ravikanth Nanduri

Mycobacterium tuberculosis Modulates Macrophage Lipid-Sensing Nuclear Receptors PPARγ and TR4 for Survival

Mycobacterium tuberculosis–macrophage interactions are key to pathogenesis and clearance of these bacteria. Although interactions between M. tuberculosis-associated lipids and TLRs, non-TLRs, and opsonic receptors have been investigated, interactions of these lipids and infected macrophage lipid repertoire with lipid-sensing nuclear receptors expressed in macrophages have not been addressed. In this study, we report that M. tuberculosis–macrophage lipids can interact with host peroxisome proliferator-activated receptor γ and testicular receptor 4 to ensure survival of the pathogen by modulating macrophage function. These two lipid-sensing nuclear receptors create a foamy niche within macrophage by modulating oxidized low-density lipoprotein receptor CD36, phagolysosomal maturation block by induction of IL-10, and a blunted innate response by alternative polarization of the macrophages, which leads to survival of M. tuberculosis. These results also suggest possible heterologous ligands for peroxisome proliferator-activated receptor γ and testicular receptor 4 and are suggestive of adaptive or coevolution of the host and pathogen. Relative mRNA expression levels of these receptors in PBMCs derived from clinical samples convincingly implicate them in tuberculosis susceptibility. These observations expose a novel paradigm in the pathogenesis of M. tuberculosis amenable for pharmacological modulation.

Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes.

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBMs ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein Giα1, as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

Mycobacterium tuberculosis Keto-Mycolic Acid and Macrophage Nuclear Receptor TR4 Modulate Foamy Biogenesis in Granulomas: A Case of a Heterologous and Noncanonical Ligand-Receptor Pair

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA–TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Background: Transcriptional modulation of IL10, a cytokine that blocks phagolysosome maturation, is not well understood. Results: This study demonstrates human IL10 gene repression by direct binding of Rev-erbα on Rev-DR2 in the proximal promoter. Conclusion: Rev-erbα binds to IL10 proximal promoter, represses expression, and impedes Mycobacterium tuberculosis in human macrophages. Significance: This study provides rationale to target Rev-erbα as a therapeutic intervention that might support host defense in tuberculosis. Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.

Nuclear Receptor Nr4a2 Promotes Alternative Polarization of Macrophages and Confers Protection in Sepsis.

Background: An understanding of the role of Nr4a2 in inflammation is needed. Results: Nr4a2 is a transcription factor that induces expression of M2 characteristic genes, and adoptive transfer of macrophages overexpressing Nr4a2 gives protection against septic mortality. Conclusion: Our data impart a new role for Nr4a2 in skewing macrophage plasticity to M2 type. Significance: Therapeutic intervention of Nr4a2 may provide a cure for inflammatory diseases. The orphan nuclear receptor Nr4a2 is known to modulate both inflammatory and metabolic processes, but the mechanism by which it regulates innate inflammatory homeostasis has not been adequately addressed. This study shows that exposure to ligands for Toll-like receptors (TLRs) robustly induces Nr4a2 and that this induction is tightly regulated by the PI3K-Akt signaling axis. Interestingly, exogenous expression of Nr4a2 in macrophages leads to their alternative phenotype with induction of genes that are prototypical M2 markers. Moreover, Nr4a2 transcriptionally activates arginase 1 expression by directly binding to its promoter. Adoptive transfer experiments revealed that increased survival of animals in endotoxin-induced sepsis is Nr4a2-dependent. Thus our data identify a previously unknown role for Nr4a2 in the regulation of macrophage polarization.

Stem Bromelain–Induced Macrophage Apoptosis and Activation Curtail Mycobacterium tuberculosis Persistence

BACKGROUND Mycobacterium tuberculosis, the causative agent of tuberculosis, has a remarkable ability to usurp its hosts innate immune response, killing millions of infected people annually. One approach to manage infection is prevention through the use of natural agents. In this regard, stem bromelain (SBM), a pharmacologically active member of the sulfhydryl proteolytic enzyme family, obtained from Ananas comosus and possessing a remarkable ability to induce the innate and acquired immune systems, is important. METHODS We evaluated SBMs ability to induce apoptosis and free-radical generation in macrophages. We also studied antimycobacterial properties of SBM and its effect on foamy macrophages. RESULTS SBM treatment of peritoneal macrophages resulted in the upregulation of proapoptotic proteins and downregulation of antiapoptotic proteins. Additionally, SBM treatment activated macrophages, curtailed the levels of free glutathione, and augmented the production of hydrogen peroxide, superoxide anion, peroxynitrite, and nitric oxide. SBM cleaves CD36 and reduced the formation of foam cells, the hallmark of M. tuberculosis infection. These conditions created an environment for the increased clearance of M. tuberculosis. CONCLUSIONS Together these data provide a mechanism for antimycobacterial activity of SBM and provide important insights for the use of cysteine proteases as immunomodulatory agents.

The Active Form of Vitamin D Transcriptionally Represses Smad7 Signaling and Activates Extracellular Signal-regulated Kinase (ERK) to Inhibit the Differentiation of a Inflammatory T Helper Cell Subset and Suppress Experimental Autoimmune Encephalomyelitis

Background: Transcriptional regulation of Smads that modulate T helper (Th) cell differentiation is not well understood. Results: Active form of vitamin D (1,25(OH)2D3) leads to VDR-RXR-Smad3-HDAC2 repressive complex on VDRE-Smad7 promoter. 1,25(OH)2D3 activates ERK. Conclusion: 1,25(OH)2D3-VDR represses Smad7 and activates ERK leading to inhibition of inflammatory T cells and EAE. Significance: TGFβ Smad and non-Smad MAPK are involved in 1,25(OH)2D3-VDR mediated inhibition of EAE. The ability of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to transcriptionally modulate Smads to inhibit Th17 differentiation and experimental autoimmune encephalomyelitis (EAE) has not been adequately studied. This study reports modulation of Smad signaling by the specific binding of the VDR along with its heterodimeric partner RXR to the negative vitamin D response element on the promoter of Smad7, which leads to Smad7 gene repression. The vitamin D receptor-mediated increase in Smad3 expression partially explains the IL10 augmentation seen in Th17 cells. Furthermore, the VDR axis also modulates non-Smad signaling by activating ERK during differentiation of Th17 cells, which inhibits the Th17-specific genes il17a, il17f, il22, and il23r. In vivo EAE experiments revealed that, 1,25(OH)2D3 suppression of EAE correlates with the Smad7 expression in the spleen and lymph nodes. Furthermore, Smad7 expression also correlates well with IL17 and IFNγ expression in CNS infiltered inflammatory T cells. We also observed similar gene repression of Smad7 in in vitro differentiated Th1 cells when cultured in presence of 1,25(OH)2D3. The above canonical and non-canonical pathways in part address the ability of 1,25(OH)2D3-VDR to inhibit EAE.

Oleic acid complex of bovine α-lactalbumin induces eryptosis in human and other erythrocytes by a Ca 2+ -independent mechanism

BACKGROUND Complexes of oleic acid (OA) with milk α-lactalbumin, received remarkable attention in view of their selective toxicity towards a spectrum of tumors during the last two decades. OA complexes of some structurally related/unrelated proteins are also tumoricidal. Erythrocytes are among the few differentiated cells that are sensitive and undergo hemolysis when exposed to the complexes. METHODS The effects of OA complex of bovine α-lactalbumin (Bovine Alpha-lactalbumin Made LEthal to Tumor cells, BAMLET) on human, goat and chicken erythrocytes on calcein leakage, phosphatidylserine exposure, morphological changes and hemolysis were studied by confocal microscopy, FACS analysis, scanning electron microscopy and measuring hemoglobin release. RESULTS Erythrocytes exposed to BAMLET undergo eryptosis-like alterations as revealed by calcein leakage, surface phosphatidylserine exposure and transformation to echinocytes at low concentrations and hemolysis when the concentration of the complex was raised. Ca(2+) was not essential and restricted the alterations when included in the medium. The BAMLET-induced alterations in human erythrocytes were prevented by the cation channel inhibitors, amiloride and BaCl2 but not by inhibitors of thiol proteases, sphingomyelinase and by the antioxidant N-acetyl cysteine. CONCLUSIONS The work shows for the first time that low concentrations of BAMLET induces eryptosis in erythrocytes by a novel mechanism not requiring Ca(2+) and hemolysis by detergent-like action by the released OA at higher concentrations. GENERAL SIGNIFICANCE The study points out to the need for a comprehensive evaluation of the toxicity of OA complexes of α-lactalbumin and other proteins towards erythrocytes and other differentiated cells before being considered for therapy.

Nuclear MEK1 Sequesters PPARγ and Bisects MEK1/ERK Signaling: A Non-Canonical Pathway of Retinoic Acid Inhibition of Adipocyte Differentiation

Uncontrolled adipogenesis and adipocyte proliferation have been connected to human comorbidities. Retinoic acid (RA) is known to inhibit adipocyte differentiation, however the underlying mechanisms have not been adequately understood. This study reports that RA acting as a ligand to RA receptors (RARs and RXRs) is not a sine qua non to the inhibition of adipogenesis. Our intriguing observation of a negative correlation between increased retinoylation and adipogenesis led us to explore retinoylated proteins in adipocytes. Exportin (CRM1) was found to be retinoylated, which in turn can affect the spatio-temporal regulation of the important signaling molecule mitogen-activated protein kinase kinase 1 (MEK1), likely by disrupting its export from the nucleus. Nuclear enrichment of MEK1 physically sequesters peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis, from its target genes and thus inhibits adipogenesis while also disrupting the MEK1-extracellular-signal regulated kinase (ERK) signaling cascade. This study is first to report the inhibition of adipocyte differentiation by retinoylation.

ONRLDB—manually curated database of experimentally validated ligands for orphan nuclear receptors: insights into new drug discovery

Orphan nuclear receptors are potential therapeutic targets. The Orphan Nuclear Receptor Ligand Binding Database (ONRLDB) is an interactive, comprehensive and manually curated database of small molecule ligands targeting orphan nuclear receptors. Currently, ONRLDB consists of ∼11 000 ligands, of which ∼6500 are unique. All entries include information for the ligand, such as EC50 and IC50, number of aromatic rings and rotatable bonds, XlogP, hydrogen donor and acceptor count, molecular weight (MW) and structure. ONRLDB is a cross-platform database, where either the cognate small molecule modulators of a receptor or the cognate receptors to a ligand can be searched. The database can be searched using three methods: text search, advanced search or similarity search. Substructure search, cataloguing tools, and clustering tools can be used to perform advanced analysis of the ligand based on chemical similarity fingerprints, hierarchical clustering, binning partition and multidimensional scaling. These tools, together with the Tree function provided, deliver an interactive platform and a comprehensive resource for identification of common and unique scaffolds. As demonstrated, ONRLDB is designed to allow selection of ligands based on various properties and for designing novel ligands or to improve the existing ones. Database URL: http://www.onrldb.org/