Defining how LPS mediates non-canonical… (F32AI200195) – Project Grant to The Trustees Of The University…

Abstract: Sepsis, which occurs from an overactive immune response to an infectious agent, is one of the most widespread and deadly illnesses in the world. The uncontrolled inflammation that ensues leads to tissue damage throughout the body, and if left untreated, can cause organ failure and become fatal. Current treatments include supportive care and the administration of antibiotics, but therapies that specifically target the inflammatory aspect of sepsis are lacking. To develop these therapeutics, a fundamental understanding of how the immune system senses pathogens is required. Currently, i study the mechanisms by which innate immune cells sense and respond to bacterial pathogens. One such mechanism is the recognition of a molecule on the outer membrane of gram- negative bacteria, called lipopolyssahride (LPS). Normally, detection of LPS by innate immune cells triggers sufficient levels of cytokines to instruct the immune system to resolve the infection and protect the host. Upon an infection of the bloodstream, however, rampant LPS sensing is thought to contribute significantly to the inflammation that ensues during sepsis in humans. Given that LPS is an actionable drug target for anti-sepsis therapeutics, my long-term research objective is to uncover what aspects of LPS are sensed by innate immune cells and how to modulate this interaction. In aim 1 of this proposal, i seek to elucidate the moieties of LPS that mediate activation of the non-canonical inflammasome pathway, which occurs in response to intracellular LPS. In this pathway, sensing of LPS leads to self-oligomerization of pro-enzymes caspase-11 (CASP11) in mice or its ortholog caspase-4 (CASP4) in humans. Once oligomerized they become active enzymes which cause cell death and inflammatory cytokine release. In aim 2, i intend to elucidate why the human non-canonical inflammasome senses a broader repertoire of LPS structures than the murine non-canonical inflammasome, enabling US to translate findings in murine studies to humans with higher efficacy. To achieve these two aims, i will employ biochemical assays that clearly demonstrate LPS-induced non-canonical inflammasome formation, cell death, and cytokine release. To delineate what component of LPS-induces this response, modified LPS molecules will be generated in aim 1, which i will deliver into macrophages to examine their capacity to induce CASP11 or CASP4 into an active inflammasome. In aim 2, i will generate chimeras of CASP11 and CASP4 to examine which region of CASP4 confers its capacity to recognize a broader repertoire of LPS structures. These aims will be performed under the supervision of sunny shin and igor brodsky, who have a breadth of knowledge in immunology and microbiology and a great track record of mentorship. Furthermore, working at the university of pennsylvania will provide a collaborative scientific environment that will propel my research forward and provide all the necessary support to successfully complete the aims set forth in this fellowship application.

Defining how LPS mediates non-canonical inflammasome activation

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