Amino Acid Sensing via General Control Nonderepressible-2 Kinase and Immunological Programming

Metabolic adaptation to the changing nutrient levels in the cellular microenvironment plays a decisive role in the maintenance of homeostasis. Eukaryotic cells are equipped with nutrient sensors, which sense the fluctuating nutrients levels and accordingly program the cellular machinery to mount an appropriate response. Nutrients including amino acids play a vital role in maintaining cellular homeostasis. Therefore, over the evolution, different species have developed diverse mechanisms to detect amino acids abundance or scarcity. Immune responses have been known to be closely associated with the cellular metabolism especially amino acid sensing pathway, which influences innate as well as adaptive immune-effector functions. Thus, exploring the cross-talk between amino acid sensing mechanisms and immune responses in disease as well as in normal physiological conditions might open up avenues to explore how this association can be exploited to tailor immunological functions toward the design of better therapeutics for controlling metabolic diseases. In this review, we discuss the advances in the knowledge of various amino acid sensing pathways including general control nonderepressible-2 kinase in the control of inflammation and metabolic diseases

Statistical design and evaluation of a propranolol HCl gastric floating tablet

The purpose of this research was to apply statistical design for the preparation of a gastric floating tablet (GFT) of propranolol HCl and to investigate the effect of formulation variables on drug release and the buoyancy properties of the delivery system. The contents of polyethylene oxide (PEO) WSR coagulant and sodium bicarbonate were used as independent variables in central composite design of the best formulation. Main effects and interaction terms of the formulation variables were evaluated quantitatively using a mathematical model approach showing that both independent variables have significant effects on floating lag time, % drug release at 1 h (D1 h) and time required to release 90% of the drug (t90). The desired function was used to optimize the response variables, each with a different target, and the observed responses were in good agreement with the experimental values. FTIR and DSC studies of the statistically optimized formulation revealed there was no chemical interaction between drug and polymer. The statistically optimized formulation released drug according to first order kinetics with a non-Fickian diffusion mechanism. Evaluation of the optimized formulation in vivo in human volunteers showed that the GFT was buoyant in gastric fluid and that its gastric residence time was enhanced in the fed but not the fasted state

Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1β (IL-1β) and provides protection from intestinal inflammation in mice. HF inhibits IL-1β through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1β mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1β production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1β is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases

HF treatment promotes IL-1β mRNA degradation by targeting it to SGs through recruitment of TIA-1/TIAR.

<p>(A) Immunoblot analysis of RBPs, TIA-1/TIAR, in the lysates of J774A.1 macrophages, left untreated or treated with varying concentrations of HF for 3-h, 6-h, and 12-h time points. β-actin was used as loading control. (B) J774A.1 macrophages were transfected with either control siRNA or TIA-1/TIAR siRNA. After 24 h, cells were either left untreated or stimulated with LPS (500 ng/ml) or LPS (500 ng/ml) plus HF (20 nM) for 6 h, ATP (5 mM) was added to the macrophage cultures for 30 min at the end of time point and were assayed for IL-1β levels in culture supernatants by ELISA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (C) IL-1β expression in the whole-cell lysates of HEK293T cells transiently expressing various combinations of plasmids (pcDNA3, pcDNA3-IL-1β, and pEYFP-TIA-1/TIAR) as indicated (top). β-actin was used as loading control. (D) RT-PCR analysis of IL-1β expression in RIP material (pull-down using TIA-1/TIAR or IgG) of lysates from J774A.1 macrophages left untreated or treated with LPS or LPS plus HF. (E) Immunoblot analysis of pro–IL-1β or pro–caspase-1 expression in macrophages treated with LPS or LPS plus HF; β–actin was used as a loading control. (F) qRT-PCR analysis of IL-1β mRNA in the RIP material from LPS-primed J774A.1 macrophages treated with the indicated concentrations of HF (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (G) qRT-PCR analysis of IL-1β mRNA in HEK293T cells transfected with constructs pCMV6-IL1β or pCMV6 IL-1β Δ3'UTR ARE, or cotransfected along with pEYFP-TIA-1/TIAR (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). Statistical significance was determined by student <i>t</i> test. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.005, ***<i>P</i> ≤ 0.0005. Data are representative of 1 of 3 independent experiments. HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IgG, immunoglobulin G; IL-1β, interleukin 1β; LPS, lipopolysaccharide; qRT-PCR, quantitative reverse transcription PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; siRNA, small interfering RNA; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.</p

HF ameliorates LPS-induced production of IL-1β in macrophages by affecting mRNA stability and processing of mature IL-1β.

<p>(A–B) IL-1β production from LPS (500 ng/ml)-primed or -unprimed BMDMs treated with different concentrations of HF or MAZ1310 (control) for 6 h. ATP (5 mM) was added to the LPS-stimulated macrophage cultures for 30 min at the end of time point (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). Statistical significance was determined by student <i>t</i> test. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.005, ***<i>P</i> ≤ 0.0005. (C) IL-1β production from LPS-primed macrophages stimulated with MSU (150 ug/ml), or ALU (200 ug/ml) for 6 h, or infected with <i>S</i>. <i>typhimurium</i> (MOI 10) in presence or absence of HF (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (D) IL-1β and caspase-1 (pro and active) expression by immunoblot analysis from LPS-primed BMDMs treated with HF as indicated; β-actin was used as loading control. (E) ROS levels detected by CM-H2DCFDA staining in macrophages treated with HF or LPS plus HF. (F, G) qRT-PCR analysis of mature IL-1β and pre–IL-1β mRNA levels in J774A.1 cells stimulated with LPS or LPS plus HF (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (H) Analysis of IL-1β mRNA levels by qRT-PCR in LPS-primed macrophages treated with Act-D for 2 h followed by HF treatment for an additional 2 h (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.005, ***<i>P</i> ≤ 0.0005 were considered statistically significant. Data are representative of 1 of 3–4 independent experiments. Act-D, actinomycin-D; ALU, aluminum hydroxide; BMDM, bone marrow–derived macrophage; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; Lys., cell lysates; MOI, multiplicity of infection; MSU, monosodium urate; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; Sup., culture supernatant.</p

HF attenuates LPS-induced IL-1β production through GCN2-dependent activation of PTR events such as riboclustering and SG formation.

<p>(A) Immunoblot analysis of GCN2 and eIF2-α phosphorylation in the lysates of macrophages treated or untreated with varying concentrations of HF for 3 h. (B) Confocal microscopy imaging of SGs, indicated by white arrows in macrophages stimulated with HF (20 nM) for 3 h; nuclei were stained with DAPI (blue). Scale bars, 10 μm. (C) Quantification of average number of SGs per cell, from 5 different fields taken from the results in panel B (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (D) Confocal microscopy imaging of SGs in WT (top) or GCN2^−/− MEFs (bottom). (E) Quantification of an average number of SGs per cell in WT or GCN2^−/− cells treated with HF (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (F, G) IL-1β (panel F) or TNF-α (panel G) levels by ELISA in the culture supernatants of WT or GCN2^−/− BMDMs primed with LPS for 3 h followed by HF treatment. ATP (5 mM) was added to the cultures for 30 min at the end of the experiment (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). *<i>P <</i> 0.05, **<i>P <</i> 0.005. (H) J774A.1 macrophages were transfected with either control siRNA or GCN2 siRNA and stimulated with LPS (500 ng/ml) or LPS plus HF (20 nM); ATP (5 mM) was added to the cultures for 30 min at the end of the experiment. IL-1β (pro and active) forms were examined in the cell lysates and culture supernatants by immunoblotting. Data are representative of 1 of 3 similar experiments. BMDM, bone marrow–derived macrophage; eIF2, eukaryotic initiation factor 2; GCN2, general control nonderepressible 2 kinase; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; Lys, cell lysates; MEF, mouse embryonic fibroblast cell; PTR, post-transcriptional reprogramming; SG, stress granule; siRNA, small interfering RNA; Sup, culture supernatant; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related; TNF, tumor necrosis factor; WT, wild-type.</p

Proposed model of the mechanism by which HF suppresses IL-1β expression.

<p>HF induces activation of the amino acid starvation sensor, GCN2, which triggers SG formation and autophagy. SG formation regulates IL-1β expression at mRNA level via translational silencers, TIA-1/TIAR and RBPs. Later, these SGs are cleared by autophagy. On the other hand, the activated autophagy process decreases ROS levels, thereby inhibiting inflammasome activation, leading to decrease in active caspase-1 and secretion of mature IL-1β. eIF2, eukaryotic initiation factor 2; GCN2, general control nonderepressible 2 kinase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; HF, Halofuginone; IL-1β, interleukin 1β; RBP, RNA-binding protein; ROS, reactive oxygen species; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.</p

HF mitigates the severity of DSS-induced colitis in mice.

<p>(A) Body weight (percentage of initial body weight) of mice (<i>n</i> = 5) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (B) Quantification of IL-1β levels by ELISA in serum samples of the indicated mice (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). **<i>P</i> ≤ 0.0015. (C) Visualization of rectal bleeding. (D) Visualization of typical colon length in control, HF-, DSS-, and DSS plus HF–treated mice. (E) Measurement of colon length (cm) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (F) Visualization of mucosal epithelium erosion and crypt loss in colon sections (HE-stained) as indicated. (G) Immunoblot analysis of IL-1β levels in the large intestine tissue samples. (H) Densitometric analysis of pro–IL-1β levels from colon tissues of mice subjected to treatments as indicated (<i>n</i> = 4) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). β-actin was used as loading control. Data are representative of 1 of 3 separate experiments. DSS, dextran sulfate sodium; HE, hematoxylin–eosin; HF, Halofuginone; IL-1β, interleukin 1β.</p

IL-1β transcripts targeted to SGs are degraded by the activation of autophagy during HF treatment.

<p>(A) Confocal microscopy imaging of LC3 punctates (indicated by white arrows) in BMDMs left untreated (media control) or treated with HF (20 nM) at time points indicated. Scale bars, 5 μm. (B) LC3 punctate counts per cell (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). (C) Immunoblot analysis of autophagy marker, LC3 in J774A.1 macrophages left untreated or treated with HF in the presence or absence of autophagy inhibitor Baf. (D) Immunoblotting of LC3 in the lysates of J774A.1 macrophages treated with LPS plus HF or LPS alone. ATP was added to the cultures for 30’ at the end of the experiment. β-actin was used as a loading control. (E) Immunoblot analysis of p62/SQSTM1 expression in J774A.1 macrophages stimulated with LPS plus ATP or LPS plus HF plus ATP in the presence or absence of autophagy inhibitor Baf (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 data</a>). (F) Immunoblot analysis of TIA-1/TIAR expression in J774A.1 macrophages treated with HF or LPS in the presence or absence of Baf (10 nM). (G–I) Quantification of IL-1β levels using ELISA in culture supernatants of macrophages stimulated with LPS (500 ng/ml) alone or LPS (500 ng/ml) plus HF (20 nM) in the presence or absence of pharmacological inhibitors of autophagy, 3-MA, Wortmannin (500 nM), or Baf (10 nM). ATP (5 mM) was added for 30 min at the end of the experiment (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). Statistical significance was determined by student <i>t</i> test. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.005. (J, K) qRT-PCR analysis of IL-1β in HEK293T cells transfected with pCMV6-IL-1β or cotransfected with pCMV6-IL-1β plus pEYFP-TIA-1/pEYFP-TIAR followed by rapamycin (100 nM) or Baf treatment (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). ***<i>P</i> ≤ 0.0005. (L) qRT-PCR analysis of IL-1β mRNA in the RIP material of LPS-primed macrophages treated with HF in presence of Wortmannin (500 nM) or CQ (25 μM) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005317#pbio.2005317.s014" target="_blank">S1 Data</a>). Data are representative of 1 of 3 independent experiments. 3-MA, 3-methyl adenine; Baf, Bafilomycin A1; BMDM, bone marrow–derived macrophage; CQ, chloroquine; HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; p62/SQSTM1, sequestosome 1; qRT-PCR, quantitative reverse transcription PCR; RIP, RNA immunoprecipitation; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.</p