microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Dissecting the Functional Impacts of Non-Coding Genetic Variation

A large proportion of the variation in traits between individuals can be attributed to variation in the nucleotide sequence of the genome. The most commonly studied traits in human genetics are related to disease and disease susceptibility. Although scientists have identified genetic causes for over 4,000 monogenic diseases, the underlying mechanisms of many highly prevalent multifactorial inheritance disorders such as diabetes, obesity, and cardiovascular disease remain largely unknown. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. In this dissertation, I evaluate the hypothesis that such non-coding variants contribute to human traits and diseases by altering the regulation of genes rather than the sequence of those genes. I will specifically focus on studies to determine the functional impacts of genetic variation associated with two related complex traits: gestational hyperglycemia and fetal adiposity. At the genomic locus associated with maternal hyperglycemia, we found that genetic variation in regulatory elements altered the expression of the HKDC1 gene. Furthermore, we demonstrated that HKDC1 phosphorylates glucose in vitro and in vivo, thus demonstrating that HKDC1 is a fifth human hexokinase gene. At the fetal-adiposity associated locus, we identified variants that likely alter VEPH1 expression in preadipocytes during differentiation. To make such studies of regulatory variation high-throughput and routine, we developed POP-STARR, a novel high throughput reporter assay that can empirically measure the effects of regulatory variants directly from patient DNA. By combining targeted genome capture technologies with STARR-seq, we assayed thousands of haplotypes from 760 individuals in a single experiment. We subsequently used POP-STARR to identify three key features of regulatory variants: that regulatory variants typically have weak effects on gene expression; that the effects of regulatory variants are often coordinated with respect to disease-risk, suggesting a general mechanism by which the weak effects can together have phenotypic impact; and that nucleotide transversions have larger impacts on enhancer activity than transitions. Together, the findings presented here demonstrate successful strategies for determining the regulatory mechanisms underlying genetic associations with human traits and diseases, and value of doing so for driving novel biological discovery.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.