Semester

Summer

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Steven Frisch

Abstract

Inflammation is an essential component of the innate immune defense against pathogens. The process of inflammation is mediated by the rapid release of intercellular mediators, which function in the activation and recruitment of cellular components of both the innate and adaptive immune system. However, dysregulation of this process, resulting in sustained inflammation is the underlying cause of a number of common pathological conditions. These include hereditary autoinflammatory disorders, gout, arthritis, asthma, and cancer. Furthermore, IL-1beta is a key component of the tumor microenvironment, which serves to recruit tumor-associated macrophages, promote angiogenesis, enhance NF-kB activation, and suppress anti-tumor immunity. The studies presented here explore novel regulatory mechanisms of IL-1beta maturation through the modulation of the inflammasome adaptor protein ASC. ASC is an intriguing protein, which participates in both inflammatory and apoptotic pathways and is frequently silenced by aberrant methylation in a large number of cancers. It is a small protein, consisting of only two protein-interaction domains, an N-terminal PYD, and a C-terminal CARD. Numerous studies have found that it is required for inflammasome assembly following activation of a number of diverse cytoplasmic NLR receptors. Therefore, regulation of this protein has broad implications for IL-1beta maturation.;In the first study, we examined the endogenous localization of ASC. Several independent research groups had found that ectopic expression of ASC results in several distinct localization patterns including nuclear, cytoplasmic, and the formation of a characteristic perinuclear aggregate termed a 'speck'. However, the subcellular distribution of endogenous ASC had never been evaluated. Therefore, we examined the subcellular localization pattern of ASC in several monocytic cell lines as well as in primary monocytes and macrophages. We discovered that in resting monocytes, ASC is localized diffusely throughout the nucleus. However, upon inflammatory stimulation of the cells, we determined that ASC was rapidly redistributed to the cytoplasm where it subsequently formed a perinuclear aggregate, reminiscent of the 'specks' observed upon overexpression of this protein. Furthermore, we determined that other core inflammasome proteins NLRP3 and caspase-1 co-localized with ASC in these aggregates, suggesting that they represent inflammasomes. Additionally, we found that nuclear export of ASC is necessary for proper inflammasome assembly and processing of IL-1beta.;In Study 2, we identified Mycoplasma sp. as a novel activator of the inflammasome. Infection with Mycoplasma sp. cause a number of organ-specific diseases in humans, and they have also been linked to a number of proinflammatory disorders. Therefore, we hypothesized that IL-beta played a role in the innate immune defense against these pathogens. We discovered that incubation of monocytes and macrophages with either live or heat-killed Mycoplasma sp. stimulated the secretion of mature IL-1beta at levels comparable to those observed with known inflammasome activators. Furthermore, we also determined that Mycoplasma infection induced the nuclear to cytoplasmic translocation of ASC, as well as the formation of perinuclear aggregates, just as we observed with known inflammasome activators in Study 1. Finally, we showed that Mycoplasma -induced release of IL-1beta was dependent upon ASC, further indicating the role of the inflammasome in this process. Based upon this data, we hypothesize that chronic infection with Mycoplasma sp. results in sustained production of IL-1beta, which leads to a prolonged inflammatory state.;In our third and final study, we identified and characterized three ASC isoforms with regards to their subcellular localization, and their ability to function as an inflammasome adaptor protein. ASCb lacks the linker domain, ASCc possesses an in-frame deletion in the PYD, which does not disrupt the linker region or the CARD, and ASCd expresses only the first 35 amino acids of the PYD, which is fused to a novel peptide. Upon ectopic expression, each isoform exhibited a different localization pattern, and none of them formed the classic perinuclear 'speck', which has become the hallmark of full-length ASCa. Upon co-expression with either NLRP3(R260W) or caspase-1, co-localization was only observed by those isoforms, which expressed a fully intact PYD or CARD, respectively. Functionally, only ASCb was able to mediate IL-1beta processing when co-expressed with NLRP3(R260W) and pro-caspase-1, although to a lesser extent, presumably because neither of its protein interaction domains were disrupted.;In summary, these studies show novel mechanisms by which IL-1beta release is regulated and induced. The discovery of novel ASC isoforms and the role its subcellular distribution plays in the regulation of IL-1beta processing provide us with new insights into how inflammasome assembly is regulated and fin-tuned. Furthermore, the nuclear to cytoplasmic redistribution of ASC provides us with a potential new therapeutic target that would prevent the release of IL-1beta without impacting the expression of ASC.

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