Browsing by Subject "Cytokine"

The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction.

This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.

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.

Herein we detail the progress made at understanding the overall process of CD8+ T-lymphocyte noncytolytic antiviral response (CNAR). This response is comprised of 3 key components, the virus, the effector cell and the target cell, each of which contribute to noncytolytic suppression. During the course of CNAR, the effector cells express antiviral factors that induce intracellular events in the target cell resulting in host-pathogen interactions that inhibit HIV-1 gene expression. The goal of this work was to clarify each step of the process of noncytolytic suppression.

The effector cell was examined to understand the regulation of antiviral factors and to construct a profile of the factors expressed during CNAR. CD8+ T-lymphocytes from HIV-1 infected individuals express unidentified factors that suppress viral replication by inhibiting HIV-1 gene expression. Understanding the regulation of these antiviral CD8+ T cell-derived factors can provide important insights into how to elicit these factors with therapeutic regimens. For a small subset of human genes, histone deacetylases (HDACs) are epigenetic regulators that condense chromatin to repress transcription. We examined the role of epigenetics in modulating the HIV-1 suppressive factors expressed by primary CD8+ T cells from subjects naturally controlling virus replication. HIV-1 suppression by CD8+ T-lymphocytes from virus controllers was reversed up to 40% by the addition of an HDAC inhibitor. Therefore, histone deacetylation within CD8+ T-lymphocytes was necessary for potent suppression of HIV-1 infection.

Blocking HDACs impairs the ability of CD8+ T-lymphocytes to repress HIV-1 transcription, demonstrating the expression of the suppressive factors is regulated by epigenetics. We used this tool to identify the potential antiviral factors that result in decreased noncytolytic suppression. Through real-time PCR analysis of 164 genes we identified 4 genes in primary CD8+ T-lymphocytes from a virus controller, and 12 genes in a CD8+ T-cell line that were greatly downregulated in response to a HDAC inhibitor. Additionally, we analyzed the chemokine and cytokine profile of these two cell types to characterize what molecules these cells secrete during CNAR. MIP-1 Beta, MIP-1 Alpha, IP-10, and MIG correlated most strongly with the magnitude of CNAR (p < 0.0001).

The response of the target cell to the antiviral factors was analyzed to better understand how CD8+ T cell antiviral factors exert suppressive activity on the HIV-1 genome in an infected cell. Noncytolytic suppression was not dependent on epigenetic changes within the target cells, as HDAC1 within the target cell was dispensable, and histone acetylation at the HIV-1 LTR remained unchanged in the presence of CD8+ T-lymphocytes.

The genetic elements within HIV-1 and the viral protein Tat were investigated to provide insight into resistance to CNAR. Two virus isolates from the same individual with contrasting sensitivities to CNAR were investigated to identify the genetic elements that confer these phenotypes. Sequence analysis of the two isolates identified mutations in the exon splicing silencers (ESS) 2 and 3 in these viruses. ESS2 and 3 are thought to control splicing of HIV-1 Tat, however levels of spliced Tat RNA levels did not differ between the two isolates. The introduction of the ESS2 mutation into a heterologous HIV-1 isolate moderately boosted resistance to CNAR, suggesting a function for the mutation apart from spliced Tat RNA levels.

In total, a comprehensive analysis of each component of CNAR is discussed here to enhance the overall understanding of the mechanisms of CNAR.

The IFN-γ/STAT1 response is an immune signaling pathway well known for its potent pro-inflammatory and anti-viral functions. However, IFN-γ/STAT1 signaling also impacts many homeostatic and pathological aspects in the central nervous system, beyond its canonical role in controlling intracellular pathogens. IFN-γ can modulate neuronal excitability, synaptic pruning, and gene expression of pathways associated with neurodevelopmental disorders, including autism spectrum disorder (ASD) and schizophrenia (SZ). Surprisingly, the IFN response was recently identified as the most highly enriched pathway in brains of individuals with ASD and SZ. Children born to mothers who are hospitalized for infection during pregnancy are at a higher risk of developing ASD, and mouse models demonstrate that elevating cytokines during embryonic neurodevelopment cause ASD-like phenotypes. While microglia are thought to be the major targets of IFNs in the brain, neurons can respond to IFNs and require physiological levels of IFN-γ for proper function. The IFN-γ/STAT1 pathway is rapidly activated then deactivated to prevent excessive inflammation; however, neurons utilize unique IFN-γ/STAT1 activation patterns, which may contribute to the non-canonical neuron-specific downstream effects. We hypothesized that pathological IFN-γ signaling in neurons leads to neuronal dysfunction and behavioral deficits through non-canonical STAT1 signaling. Using primary neuron cultures, we demonstrated that developing neurons have differential STAT1 activation downstream of physiological versus pathological IFN-γ. Physiological levels of IFN-γ caused brief and transient STAT1 activation, while high pathological levels of IFN-γ caused robust and prolonged activation of STAT1 in neurons, but not in microglia or astrocytes. To determine the effects of prolonged STAT1 activation in vivo, we developed a novel mouse model in which STAT1 signaling is prolonged in neurons. These mice displayed hyperactive behavior and neural hypoactivity, which are common comorbidities of neurodevelopmental disorders like ASD and attention deficit hyperactivity disorder (ADHD). Moreover, we demonstrated that this phenotype is neuron specific, as mice with prolonged STAT1 activation in microglia did not have behavior deficits. Our findings suggest pathological activation of the IFN-γ/STAT1 pathway contributes to neuronal dysfunction through non-canonical STAT1 activation. Overall, the IFN-γ/STAT1 pathway is critical for normal neurodevelopment and neuronal function in adulthood and provides new insight into a neuron specific neuroimmune mechanism which may contribute to the pathophysiology of neurodevelopmental disorders.

The Immunity-Related GTPases (IRG) are a family of proteins that are induced by interferon gamma and play pivotal roles in immune and inflammatory responses. IRGs ostensibly function as dynamin-like proteins that bind to intracellular membranes, and promote remodeling and trafficking of those membranes. Prior studies have shown that loss of Irgm1 in mice leads to increased lethality to bacterial infections, as well as enhanced inflammation to non-infectious stimuli; however, the mechanisms underlying these phenotypes are unclear.

In the first chapter, the role of Irgm1 in mitochondrial dynamics is examined. The structural determinants required for Irgm1's mitochondrial localization and mitochondrial fragmentation activity are delineated. In the second and third chapters, we report that uninfected Irgm1-deficient mice to display high levels of serum cytokines typifying profound autoinflammation. Similar increases in cytokine production were also seen in cultured, IFN-γ-primed macrophages that lacked Irgm1. A series of metabolic studies indicated that the enhanced cytokine production was associated with marked metabolic changes in the Irgm1-deficient macrophages, including increased glycolysis and an accumulation of long chain acylcarnitines. Cells were exposed to the glycolytic inhibitor, 2-deoxyglucose, or fatty acid synthase inhibitors to perturb the metabolic alterations, which resulted in dampening of the excessive cytokine production. These results suggest that Irgm1-deficiency drives metabolic dysfunction in macrophages in a manner that is cell autonomous and independent of infectious triggers. This may be a significant contributor to excessive inflammation seen in Irgm1-deficient mice in different contexts.