Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Urine Metabolome Dynamics Discriminate Influenza Vaccination Response.

Influenza represents a major and ongoing public health hazard. Current collaborative efforts are aimed toward creating a universal flu vaccine with the goals of both improving responses to vaccination and increasing the breadth of protection against multiple strains and clades from a single vaccine. As an intermediate step toward these goals, the current work is focused on evaluating the systemic host response to vaccination in both normal and high-risk populations, such as the obese and geriatric populations, which have been linked to poor responses to vaccination. We therefore employed a metabolomics approach using a time-course (n = 5 time points) of the response to human vaccination against influenza from the time before vaccination (pre) to 90 days following vaccination. We analyzed the urinary profiles of a cohort of subjects (n = 179) designed to evenly sample across age, sex, BMI, and other demographic factors, stratifying their responses to vaccination as "High", "Low", or "None" based on the seroconversion measured by hemagglutination inhibition assay (HAI) from plasma samples at day 28 post-vaccination. Overall, we putatively identified 15,903 distinct, named, small-molecule structures (4473 at 10% FDR) among the 895 samples analyzed, with the aim of identifying metabolite correlates of the vaccine response, as well as prognostic and diagnostic markers from the periods before and after vaccination, respectively. Notably, we found that the metabolic profiles could unbiasedly separate the high-risk High-responders from the high-risk None-responders (obese/geriatric) within 3 days post-vaccination. The purine metabolites Guanine and Hypoxanthine were negatively associated with high seroconversion (p = 0.0032, p < 0.0001, respectively), while Acetyl-Leucine and 5-Aminovaleric acid were positively associated. Further changes in Cystine, Glutamic acid, Kynurenine and other metabolites implicated early oxidative stress (3 days) after vaccination as a hallmark of the High-responders. Ongoing efforts are aimed toward validating these putative markers using a ferret model of influenza infection, as well as an independent cohort of human seasonal vaccination and human challenge studies with live virus.

Succinate dehydrogenase inversely regulates red cell distribution width and healthy life span in chronically hypoxic mice.

Increased red cell distribution width (RDW), which measures erythrocyte mean corpuscular volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increased erythrocyte numbers, hemoglobin, and RDW, while reducing longevity in male mice. Compound heterozygous knockout (hKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc, and Sdhd reduced Sdh subunit protein levels, reduced RDW, and increased healthy life span compared with WT mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the SD of MCV showed the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites showed the largest increase in succinate with elevated succinate/fumarate and succinate/oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy life span in hypoxia and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.

Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.