Gab2 and Gab3 Redundantly Suppress Colitis by Modulating Macrophage and CD8+ T-Cell Activation

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8+ T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3−/−) were generated. Gab2/3−/− mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3−/− hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo. In spontaneous disease, intestinal intraepithelial CD8+ but much fewer CD4+, T-cells from Gab2/3−/− mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8+ T-cell activation and suppress chronic colitis

Ontogeny and Polarization of Macrophages in Inflammation: Blood monocytes versus tissue macrophages.

The explosion of new information in recent years on the origin of macrophages in the steady-state and in the context of inflammation has opened up numerous new avenues of investigation and possibilities for therapeutic intervention. In contrast to the classical model of macrophage development, it is clear that tissue-resident macrophages can develop from yolk sac-derived erythromyeloid progenitors, fetal liver progenitors and bone marrow-derived monocytes. Under both homeostatic conditions and in response to pathophysiological insult, the contribution of these distinct sources of macrophages varies significantly between tissues. Furthermore, while all of these populations of macrophages appear to be capable of adopting the polarized M1/M2 phenotypes, their respective contribution to inflammation, resolution of inflammation and tissue repair remains poorly understood and is likely to be tissue- and disease-dependent. A better understanding of the ontology and polarization capacity of macrophages in homeostasis and disease will be essential for the development of novel therapies that target the inherent plasticity of macrophages in the treatment of acute and chronic inflammatory disease

Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans

Strains, plasmids, primers and RNAs used in this study.

<p>Strains, plasmids, primers and RNAs used in this study.</p

expression of ICT1 partially complements the growth defect of δ<i>ssra</i> cells.

<p>Growth of wild-type cells, Δ<i>SSRA</i> cells, or Δ<i>SSRA</i> cells expressing ArfB or ICT1 from a plasmid was monitored during exponential phase. The average doubling times (± standard deviation) from ≥3 experiments are shown.</p

ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.

<p>In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.</p

<i>c</i>. <i>crescentus</i> ArfB rescues human cells from ICT1 silencing.

<p>Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.</p

ICT1 and ArfB share conserved residues that are required for release activity.

<p>Clustal Omega alignment of human ICT1 and ArfB proteins from <i>E</i>. <i>COLI</i> and <i>C</i>. <i>CRESCENTUS</i>. Blue stars indicate residues required for ≥ 60% ICT1 peptidyl-tRNA hydrolysis activity on non-stop ribosomes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005964#pgen.1005964.ref022" target="_blank">22</a>]. Red stars indicate residues required for ≥ 60% <i>E</i>. <i>COLI</i> ArfB hydrolysis activity on non-stop ribosomes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005964#pgen.1005964.ref022" target="_blank">22</a>]. The N-terminal extension of ICT1 contains the mitochondrial localization signal. The remaining 143 C-terminal residues, thought to constitute the active portion of ICT1, share 26% sequence identity with <i>C</i>. <i>CRESCENTUS</i> ArfB.</p

ICT1 ribosome release activity supports viability in <i>c</i>. <i>crescentus</i>.

<p>A co-transduction experiment was used to test whether ICT1 complements the synthetic lethal phenotype of deleting <i>ARFB</i> and <i>SSRA</i>. (A) Cartoon depicting the co-transduction experiment and predicted frequency of the outcomes if <i>ARFB</i> were not essential. (B) Column graph indicating the average co-transduction frequency from 3 independent experiments, with error bars indicating the standard deviation.</p

Metabolic Profiling Reveals Aggravated Non-Alcoholic Steatohepatitis in High-Fat High-Cholesterol Diet-Fed Apolipoprotein E-Deficient Mice Lacking Ron Receptor Signaling

Non-alcoholic steatohepatitis (NASH) represents the progressive sub-disease of non-alcoholic fatty liver disease that causes chronic liver injury initiated and sustained by steatosis and necroinflammation. The Ron receptor is a tyrosine kinase of the Met proto-oncogene family that potentially has a beneficial role in adipose and liver-specific inflammatory responses, as well as glucose and lipid metabolism. Since its discovery two decades ago, the Ron receptor has been extensively investigated for its differential roles on inflammation and cancer. Previously, we showed that Ron expression on tissue-resident macrophages limits inflammatory macrophage activation and promotes a repair phenotype, which can retard the progression of NASH in a diet-induced mouse model. However, the metabolic consequences of Ron activation have not previously been investigated. Here, we explored the effects of Ron receptor activation on major metabolic pathways that underlie the development and progression of NASH. Mice lacking apolipoprotein E (ApoE KO) and double knockout (DKO) mice that lack ApoE and Ron were maintained on a high-fat high-cholesterol diet for 18 weeks. We observed that, in DKO mice, the loss of ligand-dependent Ron signaling aggravated key pathological features in steatohepatitis, including steatosis, inflammation, oxidation stress, and hepatocyte damage. Transcriptional programs positively regulating fatty acid (FA) synthesis and uptake were upregulated in the absence of Ron receptor signaling, whereas lipid disposal pathways were downregulated. Consistent with the deregulation of lipid metabolism pathways, the DKO animals exhibited increased accumulation of FAs in the liver and decreased level of bile acids. Altogether, ligand-dependent Ron receptor activation provides protection from the deregulation of major metabolic pathways that initiate and aggravate non-alcoholic steatohepatitis