Our translational research program studies intermediary metabolism and the immune system with a focus on inborn errors of metabolism. Intermediary metabolism is critical for the activation and differentiation of T-cells. Abnormalities in these pathways may lead to defects in effector and memory T-cell functions. Using a combination of mouse genetics, metabolomics, fluxomics, molecular and cell biology, and mouse models of inborn errors of metabolism, our goals are to understand how intermediary metabolism contributes to the normal function of cells in the immune system and their dysfunction in inborn errors of metabolism. This immune dysfunction may be primary or secondary. Genes involved in intermediary metabolism may be expressed in T-cells. In addition, intoxication phenotypes (e.g., acidosis or hyperammonemia) may also precipitate immune dysfunction. We are particularly interested in how these pathways affect responses to infectious diseases and immunization. In addition, we also focus on disruptions of hepatic metabolism due to activation of the immune system systemically and locally. Hepatic metabolic decompensations in inborn errors of metabolism (e.g., hyperammonemia, acidosis, hypoglycemia) are most often precipitated by infection. Specifically our aims are: 1) To understand how intermediary metabolism affects T-cell development, differentiation and function. A special focus is placed in understanding how metabolic enzyme defects and intoxication phenotypes in inborn errors of metabolism affect T-cell effector and memory cell differentiation and function in response to immunization and infection. This work is being pursued in the laboratory with mouse models as well as affected patients in the NIH clinical center. 2) To understand how immune activation systemically or locally can precipitate hepatic metabolic dysfunction in inborn errors of metabolism. Our current work focuses on modeling respiratory viral infection-induced hepatopathy and resultant hyperammonemia in mouse models of urea cycle disorders. We are working on the mechanisms of cytokine and viremia induced urea cycle dysfunction. Research: Intermediary metabolism and T-cells: Over the past year we have characterized the role of the amino acids citrulline and arginine in T-cell function. For T-cells, arginine is essential for T-cell proliferation and maintenance of the T-cell receptor. In T-cells deprived of arginine, the CD3 zeta chain and T-cell receptor are downregulated from the cell surface reducing proliferation. Using stable isotopes, the fate ultimate fate of arginine so far seems to be protein and polyamine synthesis. These pathways are currently being dissected out further. During arginine deficiency states, such as during an infection, cells may utilize citrulline by upregulating argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL) to make arginine. ASS1 and ASL are also enzymes of the hepatic urea cycle. This implies that individuals with urea cycle disorders due to ASS1 (citrullinemia) or ASL (argininosuccinic aciduria) deficiencies may have a conditional immune defect under low arginine conditions. To answer these questions, we used a mouse model of citrullinemia. These animals are unable to convert citrulline to arginine and have hypercitrullinemia and hyperammonemia. Pathologic studies revealed that these mice had T and B lymphopenia, small spleens, and absent mesenteric lymph nodes. Since most of the animals die by 3 weeks of age, in conjunction with the Venditti laboratory, we developed a liver-targeted gene replacement therapy approach. This approach would also allow us to study cell autonomous ASS1 defects. To date, we have rescued several animals, which still retain the ASS1 defect in their T-cells. We are currently in the process of evaluating T-cell function using influenza A immunization and natural infection approaches. To address the effects of systemic intoxication with ammonia on immune function, we are working with another model of a urea cycle disorder, the spf-ash mouse (ornithine transcarbamylase deficiency (OTC)). These mice are hyperammonemic at baseline. Immunization studies against influenza thus far have demonstrated reduced vaccine efficacy against natural infection. We are continuing to characterize the nature of this defect, concentrating on aspects of ammonia intoxication of T-cells. To accomplish this, we are characterizing a new model of OTC deficiency, the spf-j, which will be published by our lab shortly. The advantage of this model over previous models is that the mouse is also hyperammonemic but on a clean B6 background unlike its predecessor. These basic studies have extended to the NIH Clinical Center. Our protocol titled the NIH UNI Study: Urea Cycle Disorders, Nutrition and Immunity is up and running and actively recruiting patients. In this protocol we evaluate the development of adaptive immunity to influenza and Hepatitis A vaccines. Immune activation and hepatic intermediary metabolic dysfunction: Our second main area of work involves exacerbation of hepatic metabolic diseases by activation of the immune system. One of the most serious consequences of Urea Cycle Disorder (UCD) is acute hyperammonemia (HA), which may be caused by dietary indiscretion (i.e. high protein intake), dietary deficiency resulting in enhanced catabolism, and acute infection. Acute HA is caused most often by infectious precipitants. There is also a perception that the acute HA that develops is clinically different from other precipitants: higher plasma ammonias of longer duration. Using clinical data from the Rare Disease Clinical Research Network sponsored prospective longitudinal study of the Urea Cycle Disorders Consortium, we were able to show that acute HA due to infection is a distinct clinical entity and displays markers of increased morbidity. This manuscript is in preparation. Although enhanced catabolism is present during dietary deficiency as well as acute infection, we hypothesized that immune activation with inflammatory cytokine release may have more direct effects on hepatic urea cycle function. In support of this idea, we performed in vitro studies in primary human hepatocytes from controls and patients with UCD, which demonstrated deleterious effects of inflammatory cytokines on ammonia metabolism. To examine this mechanism in vivo, we developed a model system of acute HA due to infection using two well-defined models, mouse-adapted influenza A/PR/8 and the spf-ash mouse, a model of OTC deficiency. Via fluxomic and targeted metabolomic studies, we demonstrated that influenza infection results in unique perturbations in the disposal of ammonia and the availability of anaplerotic intermediates of the urea cycle. The clinical significance and translational importance of these findings are highlighted in the current management of acute HA in UCD. Regardless of the acute HA precipitant, the medical management strategy is the same: cut protein intake for 1-2 days, and provide high caloric intake. These measures are not always successful. Our findings provide novel insight into the metabolic pathophysiology behind acute infection, provides evidence for the use of alternative pathways of ammonia disposal in the spf-ash mouse and the possibility of applying anaplerotic intermediates or immune modifiers. This suggests that the ubiquitously applied treatment strategy of reversing catabolism during acute HA due to infection could be augmented. This work has resulted in a manuscript that is at present submitted for review. Future directions for this work include dissecting the molecular and metabolic events behind urea cycle inhibition. We will focus on cytokine signaling in hepatocytes and links to metabolism as well as viremia induced hepatopathy.
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