Semester

Spring

Date of Graduation

2023

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Not Listed

Committee Chair

Salik Hussain

Committee Member

Stanley Hileman

Committee Member

Sharan Bobbala

Abstract

Introduction: The regulation of inflammation at the cellular and molecular level is a crucial aspect of homeostasis that is often overlooked, and most treatment options are limited to antibiotics and symptom management drugs. The development of novel molecular-targeted therapy options downstream of pathogen recognition receptors will fulfill the unmet need for therapy to effectively regulate excessive inflammation. NLRX1 is a unique NOD-like receptor that is anti-inflammatory in nature; it has been shown to negatively regulate the NF-kB pathway and has been involved in the pathology of a plethora of inflammatory disorders. This makes NLRX1 an emerging therapeutic molecular target for the regulation of inflammation. We investigated here the effects of NX-13, an NLRX1 agonist, in alleviating LPS-induced inflammation in various cell types in-vitro. Furthermore, in-vivo we identified a novel potential therapeutic effect of NX-13 in mitigating LPS-induced lung function decline and airway hyperresponsiveness.

Methods: The safety of NX-13 treatment was assessed in-vitro using A549 and RAW 264.7 cells. Cells were treated with various doses of NX-13 to establish a safe dose that does not negatively impact cellular viability nor induce toxic effects. Lipopolysaccharide (LPS) was utilized to induce inflammation and toxicity. Pre-treatment with NX-13 was used to evaluate its therapeutic effects in alleviating the biological activity of inflammatory inducers in human and murine macrophages in-vitro and ex-vivo. Oropharyngeal administration of NX-13 in adult C57BL/6J mice were also used to assess its in-vivo efficacy in alleviating lung inflammation, toxicity, and injury. Lung function and airway responsiveness were assessed using the FlexiVent forced oscillation technique (FOT), and negative pressure-driven forced expiration (NPFE) methods. Bronchoalveolar lavage fluid (BALF) was collected, and total cells, macrophages, and neutrophils were quantified. The biological activity of inflammatory mediators with or without the treatment of NX-13 was assessed by measuring ROS production and the release of pro-inflammatory cytokines at the mRNA and protein levels.

Results: Cellular health and viability assays demonstrated that in-vitro treatment with NX-13 up to 500nM does not induce any cytotoxic effects. Therapeutically, in-vitro delivery of NX-13 was capable of alleviating LPS-induced ROS production, mRNA abundance and protein content of pro-inflammatory cytokines, and the activation of NF-κB. Furthermore, in-vivo delivery of NX-13 by oropharyngeal aspiration briefly prior to acute exposure to LPS significantly reduced lung inflammation and air-blood barrier disruption. Furthermore, there was a trending amelioration in LPS-induced lung function decline and airway hyperresponsiveness.

Conclusion: This data demonstrates the potential of NX-13 in therapeutically targeting NLRX1 to alleviate LPS-induced inflammation, toxicity, and pulmonary function decline. This study paves a foundational path for various other studies to investigate molecular-targeted therapies using NX-13 or similar therapeutic agents to mitigate the severity of chronic inflammatory disorders.

Embargo Reason

Publication Pending

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Available for download on Friday, April 26, 2024

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