Effects of Bronchial Segmental Endotoxin Instillation in Humans

Defining the mechanisms that limit tissue injury during acute inflammation may provide the basis for preventing secondary organ dysfunction during serious infections. We plan to use the human endotoxin lung challenge model to study mechanisms associated with the initiation and resolution of lung inflammation. Endotoxin (LPS), an outer membrane component of Gram-negative bacteria, is a major microbial factor that mediates inflammation associated with serious infections. For over fifty years, endotoxin has been administered to humans as a challenge agent in order to understand basic mechanisms of inflammation, to provide a proof of principle for pharmacologic interventions (e.g., anti-cytokines, corticosteroids), or as an adjuvant for immunotherapy of malignancies. We previously developed a human model to study the initiation and resolution of inflammation in a lung segment following endotoxin bronchial instillation. The inflammatory response was limited to the challenged segment and had minimal associated systemic responses. After direct instillation into a lung segment, endotoxin elicits an inflammatory response characterized by the local production of inflammatory mediators, proteins and increases in cellularity. The predominate cell species found in the bronchoalveolar lavage (BAL) of the challenged segment changes from a neutrophil influx at 2 - 6 hours to an influx of monocytes and lymphocytes at 24 and 48 hours following endotoxin challenge. At 6 hours, BAL pro-inflammatory activity (i.e., tumor necrosis factor bioactivity and induction of intercellular adhesion molecule-1 on reporter cells) is present and absent at 24 and 48 hours post endotoxin. In addition, unique subsets of lymphocytes are present in the lung during this inflammatory response. We have recently shown that non-cellular microRNAs (miRNAs) are present in archived BAL from normal healthy volunteer airways and are differentially expressed 6 hours after endotoxin-induced acute lung inflammation. miRNA are single stranded non-coding RNA that mediate posttranscriptional regulation of gene expression. Some extracellular miRNA species may modulate local and distant cell processes. miRNA can activate Toll-like receptors (TLRs) and have been described as blood biomarkers of different disease states. However, the specific role of extracellular or secreted miRNAs in the lung is poorly defined. Our research plan is designed to study the roles of miRNA in acute lung inflammation by investigating the changes in miRNA signatures found in BAL and lung cells from healthy volunteers at baseline and at 6, 24, or 48 hours after endotoxin segmental lung challenge. A panel of flow cytometry cell surface markers will be performed on blood and BAL cells and intracellular and secreted miRNA will be isolated from specific cell species (i.e., neutrophils, monocytes, macrophages, and lymphocytes). We hypothesize that miRNA will have a role in modulating the initiation and resolution of inflammation.