Vemika Chandra

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.

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.

Specific molten globule conformation of stem bromelain at alkaline pH.

Stem bromelain (SBM), a therapeutic protein, is rapidly absorbed across the gut epithelium. Because SBM encounters an alkaline pH at its principal site of absorption, we investigated the alkaline-induced denaturation of SBM. From pH 7 to 10, the proteins secondary structure remained the same, although a slight loss of tertiary structure was observed. Above pH 10, there was a significant and irreversible loss of secondary and tertiary structure. At pH 10, SBM showed enhanced tryptophan fluorescence, however, the number of accessible tryptophans remained the same. The thermodynamics of temperature transition at pH 7 and 10 were strikingly different, with the former showing a two-phase transition endotherm, and the latter a broad non-two-state transition. At pH 10, SBM showed a significant increase in 8-anilino-1-naphthalene-sulfonate binding relative to the native state, suggestive of a specific molten globule (SMG) state. These studies suggest a distinct conformational rearrangement in SBM, at the proteins isoelectric point.

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.

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the proteins isoelectric point.

Transcription factors and cognate signalling cascades in the regulation of autophagy

Autophagy is a process that maintains the equilibrium between biosynthesis and the recycling of cellular constituents; it is critical for avoiding the pathophysiology that results from imbalance in cellular homeostasis. Recent reports indicate the need for the design of high‐throughput screening assays to identify targets and small molecules for autophagy modulation. For such screening, however, a better understanding of the regulation of autophagy is essential. In addition to regulation by various signalling cascades, regulation of gene expression by transcription factors is also critical. This review focuses on the various transcription factors as well as the corresponding signalling molecules that act together to translate the stimuli to effector molecules that up‐ or downregulate autophagy. This review rationalizes the importance of these transcription factors functioning in tandem with cognate signalling molecules and their interfaces as possible therapeutic targets for more specific pharmacological interventions.

Discovery of Mycobacterium tuberculosis α-1,4-Glucan Branching Enzyme (GlgB) Inhibitors by Structure- and Ligand-based Virtual Screening

Background: M. tuberculosis GlgB is essential for the biosynthesis of branched glucan and modulates pathogenesis and survival. Results: Two novel small molecules demonstrated significant inhibition of M. tuberculosis GlgB enzyme activity, growth and survival. Conclusion: Small molecules with diverse scaffolds but similar three-dimensional-structures show a similar biological effect. Significance: Deriving scaffolds from docking and similarity search is a successful design strategy for difficult targets. GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.

NR1D1 ameliorates Mycobacterium tuberculosis clearance through regulation of autophagy

NR1D1 (nuclear receptor subfamily 1, group D, member 1), an adopted orphan nuclear receptor, is widely known to orchestrate the expression of genes involved in various biological processes such as adipogenesis, skeletal muscle differentiation, and lipid and glucose metabolism. Emerging evidence suggests that various members of the nuclear receptor superfamily perform a decisive role in the modulation of autophagy. Recently, NR1D1 has been implicated in augmenting the antimycobacterial properties of macrophages and providing protection against Mycobacterium tuberculosis infection by downregulating the expression of the IL10 gene in human macrophages. This antiinfective property of NR1D1 suggests the need for an improved understanding of its role in other host-associated antimycobacterial pathways. The results presented here demonstrate that in human macrophages either ectopic expression of NR1D1 or treatment with its agonist, GSK4112, enhanced the number of acidic vacuoles as well as the level of MAP1LC3-II, a signature molecule for determination of autophagy progression, in a concentration- and time-dependent manner. Conversely, a decrease in NR1D1 in knockdown cells resulted in the reduced expression of lysosomal-associated membrane protein 1, LAMP1, commensurate with a decrease in the level of transcription factor EB, TFEB. This is indicative of that NR1D1 may have a regulatory role in lysosome biogenesis. NR1D1 being a repressor, its positive regulation on LAMP1 and TFEB is suggestive of an indirect byzantine mechanism of action. Its role in the modulation of autophagy and lysosome biogenesis together with its ability to repress IL10 gene expression supports the theory that NR1D1 has a pivotal antimycobacterial function in human macrophages.