Redefining the Role of Lymphotoxin Beta Receptor in the Maintenance of Lymphoid Organs and Immune Cell Homeostasis in Adulthood.

Lymphotoxin beta receptor (LTßR) is a promising therapeutic target in autoimmune and infectious diseases as well as cancer. Mice with genetic inactivation of LTßR display multiple defects in development and organization of lymphoid organs, mucosal immune responses, IgA production and an autoimmune phenotype. As these defects are imprinted in embryogenesis and neonate stages, the impact of LTßR signaling in adulthood remains unclear. Here, to overcome developmental defects, we generated mice with inducible ubiquitous genetic inactivation of LTßR in adult mice (iLTßRΔ/Δ mice) and redefined the role of LTßR signaling in organization of lymphoid organs, immune response to mucosal bacterial pathogen, IgA production and autoimmunity. In spleen, postnatal LTßR signaling is required for development of B cell follicles, follicular dendritic cells (FDCs), recruitment of neutrophils and maintenance of the marginal zone. Lymph nodes of iLTßRΔ/Δ mice were reduced in size, lacked FDCs, and had disorganized subcapsular sinus macrophages. Peyer`s patches were smaller in size and numbers, and displayed reduced FDCs. The number of isolated lymphoid follicles in small intestine and colon were also reduced. In contrast to LTßR-/- mice, iLTßRΔ/Δ mice displayed normal thymus structure and did not develop signs of systemic inflammation and autoimmunity. Further, our results suggest that LTßR signaling in adulthood is required for homeostasis of neutrophils, NK, and iNKT cells, but is dispensable for the maintenance of polyclonal IgA production. However, iLTßRΔ/Δ mice exhibited an increased sensitivity to C. rodentium infection and failed to develop pathogen-specific IgA responses. Collectively, our study uncovers new insights of LTßR signaling in adulthood for the maintenance of lymphoid organs, neutrophils, NK and iNKT cells, and IgA production in response to mucosal bacterial pathogen.

Addiction to Coupling of the Warburg Effect with Glutamine Catabolism in Cancer Cells.

Metabolic reprogramming is critical to oncogenesis, but the emergence and function of this profound reorganization remain poorly understood. Here we find that cooperating oncogenic mutations drive large-scale metabolic reprogramming, which is both intrinsic to cancer cells and obligatory for the transition to malignancy. This involves synergistic regulation of several genes encoding metabolic enzymes, including the lactate dehydrogenases LDHA and LDHB and mitochondrial glutamic pyruvate transaminase 2 (GPT2). Notably, GPT2 engages activated glycolysis to drive the utilization of glutamine as a carbon source for TCA cycle anaplerosis in colon cancer cells. Our data indicate that the Warburg effect supports oncogenesis via GPT2-mediated coupling of pyruvate production to glutamine catabolism. Although critical to the cancer phenotype, GPT2 activity is dispensable in cells that are not fully transformed, thus pinpointing a metabolic vulnerability specifically associated with cancer cell progression to malignancy.

Transformation with oncogenic Ras and the simian virus 40 T antigens induces caspase-dependent sensitivity to fatty acid biosynthetic inhibition.

UNLABELLED: Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras(12V), affect fatty acid metabolism. Our results indicate that SV40/Ras(12V)-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras(12V)-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras(12V). Similar to what was observed in the transformed fibroblasts, the Ras(12V)-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. IMPORTANCE: Viral oncoproteins and cellular mutations drive the transformation of normal cells to the cancerous state. These oncogenic alterations induce metabolic changes and dependencies that can be targeted to kill cancerous cells. Here, we find that the cellular transformation resulting from combined expression of the SV40 early region with an oncogenic Ras allele is sufficient to induce cellular susceptibility to fatty acid biosynthetic inhibition. Inhibition of fatty acid biosynthesis in these cells resulted in programmed cell death, which could be rescued by supplementing the medium with nonsaturated fatty acids. Similar results were observed with the expression of oncogenic Ras in nontransformed breast epithelial cells. Combined, our results suggest that specific oncogenic alleles induce metabolic dependencies that can be exploited to selectively kill cancerous cells.

Citrobacter rodentium-induced colitis: A robust model to study mucosal immune responses in the gut.

Citrobacter rodentium is a natural mouse pathogen which reproducibly infects mice and causes intestinal disease. The C. rodentium model of infection is very useful for investigating host-pathogen immune interactions in the gut, and can also be used to understand the pathogenesis of several important human intestinal disorders, including Crohn's disease, ulcerative colitis, dysbiosis and colon tumorigenesis. Both innate and adaptive immune responses play a critical role in protection against C. rodentium. Here, we summarize the role of immune components in protection against C. rodentium and describe techniques for the analysis of innate and adaptive mucosal immune responses, including setting up the infection, analysis of colonic hyperplasia and bacterial dissemination, evaluation of antibody responses, and purification and analysis of intestinal epithelial and lymphoid cells.

Distinct domains within the human cytomegalovirus U(L)26 protein are important for wildtype viral replication and virion stability.

The human cytomegalovirus (HCMV) U(L)26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the U(L)26 protein that contribute to viral infection, we created a panel of site-directed U(L)26 mutant viruses and assessed their impact on phenotypes attributed to U(L)26. We find that the C-terminal 38 amino acids of the U(L)26 protein are absolutely necessary for U(L)26 function. A stop-insertion mutant that produced a truncated U(L)26 protein lacking this region behaved identically to U(L)26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of U(L)26 packaged into the virion resulting in reduced delivery of U(L)26 to newly infected cells. Further, this C-terminal truncated U(L)26 exhibited substantially reduced nuclear localization compared to wildtype U(L)26. Translation of U(L)26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of U(L)26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of U(L)26, are also important for the function of the U(L)26 protein. A viral mutant that produces only the short isoform of U(L)26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short U(L)26 isoform in the presence of the long U(L)26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the U(L)26 protein that contribute to HCMV infection.

Human cytomegalovirus induces the activity and expression of acetyl-coenzyme A carboxylase, a fatty acid biosynthetic enzyme whose inhibition attenuates viral replication.

We have previously reported that human cytomegalovirus (HCMV) infection induces large-scale changes to host cell glycolytic, nucleic acid, and phospholipid metabolism. Here we explore the viral mechanisms involved in fatty acid biosynthetic activation. Our results indicate that HCMV targets ACC1, the rate-limiting enzyme of fatty acid biosynthesis, through multiple mechanisms. HCMV infection was found to activate ACC1 expression, increasing the abundance of both ACC1 mRNA and protein. Viral gene expression but not viral DNA replication was found to be necessary for HCMV-mediated induction of ACC1 levels. HCMV infection was also found to increase the proteolytic processing of SREBP-2, a transcription factor whose proteolytic cleavage is known to activate a variety of phospholipid metabolic genes. Processing of SREBP-2 was found to be dependent on mTOR activity; pharmaceutical inhibition of mTOR blocked HCMV-induced SREBP-2 processing and prevented the induction of fatty acid biosynthesis and ACC1 expression. Independent of the increases in ACC1 expression, HCMV infection also induced ACC1's enzymatic activity. Inhibition of ACC1 through either RNA interference (RNAi) or inhibitor treatment was found to attenuate HCMV replication, and HCMV replication was sensitive to ACC1 inhibition even at the later stages of infection, suggesting a late role for fatty acid biosynthesis during HCMV replication. These findings indicate that HCMV infection actively modulates numerous functional aspects of a key metabolic regulatory enzyme that is important for high-titer viral replication.

In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine.

The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia-reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min occlusion plus 2 or 24 h reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 h reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.