Dedifferentiation Of Cytotoxic Lymphocytes Into Central Memory Cd8+ T Cells: Lessons From Antiviral T Stem Cells On The Architecture Of Aging & Immunotherapy

Startling advances in biological gerontology coupled with the ongoing, global demographic transition to older populations have intensified humankind’s ancient quest to understand the nature of aging. This dissertation explores mechanisms of mortality through the prism of immunology. To effect defense against pathogens both exogenous and endogenous, the adaptive immune system leverages the tremendous proliferative capacity with which it is endowed to generate terminally differentiated lymphocytes that potently eliminate or suppress threats to organismal health. This proliferative capacity derives from the integrity of genomically encoded information, and, while considerable, is not infinite: it can be depleted with age and following repeated rounds of antigen-driven proliferation, for example, when driven by recurring or persisting microbial infections, or cancer. In the studies detailed herein, genome integrity homeostasis was probed during the immune response to viral infection. CD8+ T cells were observed to experience significant DNA damage in the course of their attempts to control viral replication. Multiparametric flow cytometry identified a rare population of antigen-specific T stem cells that might represent an evolutionary strategy to minimize genotoxicity. The programming, metabolic profile, anatomic localization, proliferative capacity, and ontogeny of these T stem cells were analyzed in relation to previously established effector, memory precursor, and central memory populations. Developmentally, T stem cells were able to self-renew and both give rise to abundant cytotoxic effector CD8+ T cells in the presence of antigen as well as contribute to the pool of long-term central memory CD8+ T cells following antigen clearance. Remarkably, partially-differentiated transit-amplifying effector CD8+ T cells also contributed to long-term central memory, in a process of apparent dedifferentiation. Programmatically, T stem cells were endowed with superior expression of multiple genome maintenance and repair activities, including heightened responsiveness to DNA strand breaks, telomerase expression, and suppression of potentially mutagenic transposition by the ancient LINE-1 retrotransposon. Together, these features suggest that this characteristic of amplified genome integrity surveillance may be a fundamental feature of somatic stem cells broadly, and important for the long-term maintenance of antigen-experienced T cell populations in particular. Additionally, despite relative proliferative quiescence, T stem cells unexpectedly manifested intense signaling flux. This was associated with robust expression of a panoply of both stimulatory and inhibitory cell surface receptors, including PD-1. Intensified signaling consequent to genetic ablation of inhibitory receptors compromised T stem cell viability during acutely-resolving viral infections. These findings hold significant implications for basic understanding of pathogen-driven peripheral T cell differentiation, formation of long-term immunological memory, and aging; and for the design of therapeutic, prophylactic, and diagnostic applications intended to further the cause of maximizing healthy human lifespan

miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

Excessive or inappropriate activation of the immune system can be deleterious to the organism, warranting multiple molecular mechanisms to control and properly terminate immune responses. MicroRNAs (miRNAs), ~22-nt-long noncoding RNAs, have recently emerged as key posttranscriptional regulators, controlling diverse biological processes, including responses to non-self. In this study, we examine the biological role of miR-146a using genetically engineered mice and show that targeted deletion of this gene, whose expression is strongly up-regulated after immune cell maturation and/or activation, results in several immune defects. Collectively, our findings suggest that miR-146a plays a key role as a molecular brake on inflammation, myeloid cell proliferation, and oncogenic transformation

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Chromosomal clustering of genes controlled by the aire transcription factor

Autoimmune regulator (aire) is a transcription factor that controls the self-reactivity of the T cell repertoire. Although previous results indicate that it exerts this function in part by promoting ectopic expression of a battery of peripheral-tissue antigens in epithelial cells of the thymic medulla, recent data argue for additional roles in negative selection of thymocytes by medullary cells. As one approach to exploring such roles, we performed computational analyses of microarray data on medullary RNA transcripts from aire-deficient versus wild-type mice, focusing on the genomic localization of aire-controlled genes. Our results highlight this molecule's transcriptional activating and silencing roles and reveal a significant degree of clustering of its target genes. On a local scale, aire-regulated clusters appeared punctate, with aire-controlled and aire-independent genes often being interspersed. This pattern suggests that aire's action may not be a simple reflection of the wide action of a chromatin remodeling enzyme. Analysis of the identity of certain of the clustered genes was evocative of aire's potential roles in antigen presentation and the coordination of intrathymic cell migration: for example, major histocompatibility complex class I and class II gene products and certain chemokine genes are targets of aire-regulated transcription

Integrative Deep Sequencing of the Mouse Lung Transcriptome Reveals Differential Expression of Diverse Classes of Small RNAs in Response to Respiratory Virus Infection

We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection

Altered expression of microRNAs in the myocardium of rats with acute myocardial infarction

<p>Abstract</p> <p>Background</p> <p>MicroRNAs(miRNAs) are important cellular components and their dysfunction is associated with various diseases. Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases. Although several miRNAs are reported to be associated with AMI, more novel miRNAs are needed to further investigate and improve certainty</p> <p>Methods</p> <p>We applied a well-established acute myocardial infarction rat model and performed miRNAs microarray experiments upon the myocardium tissue of rats with AMI and under sham control. We identified the differentially expressed miRNAs and analyzed the function of miRNA targets, transcription factors, and host genes based on bioinformatics.</p> <p>Results</p> <p>As a result, the levels of expression of seventeen miRNAs significantly deregulated, of which four miRNAs were further validated by qRT-PCR. In addition, we observed that the transcription factors, targets, and host genes of these deregulated miRNAs are enriched in cardiovascular-related functions.</p> <p>Conclusion</p> <p>We found that the miRNAs expression level altered in rats with AMI and differentially expressed miRNAs may be novel biomarkers of AMI.</p

AIRE activated tissue specific genes have histone modifications associated with inactive chromatin

The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes

Expression profile of microRNAs in young stroke patients

10.1371/journal.pone.0007689PLoS ONE41

Differential Expression Profile and Genetic Variants of MicroRNAs Sequences in Breast Cancer Patients

The technology available for cancer diagnosis and prognosis is not yet satisfactory at the molecular level, and requires further improvements. Micro RNAs (miRNAs) have been recently reported as useful biomarkers in diseases including cancer. We performed a miRNA expression profiling study using peripheral blood from breast cancer patients to detect and identify characteristic patterns. A total of 100 breast cancer patients and 89 healthy patients were recruited for miRNA genotyping and expression profiling. We found that hs-miR-196a2 in premenopausal patients, and hs-miR-499, hs-miR-146a and hs-miR-196a2 in postmenopausal patients, may discriminate breast cancer patients from healthy individuals. In addition, we found a significant association between two microRNA polymorphisms (hs-miR-196a2 and hs-miR-499) and breast cancer risk. However, no significant association between the hs-miR-146a gene and breast cancer risk was found. In summary, the study demonstrates that peripheral blood miRNAs and their expression and genotypic profiles can be developed as biomarkers for early diagnosis and prognosis of breast cancer