Pulmonary Effects of Bronchial Segmental Endotoxin Instillation in Humans

我们之前开发了一个人体模型来研究内毒素支气管灌注后肺段炎症的发生和消退。炎症反应仅限于受损节段，并有最小的相关全身反应。在直接注入肺段后，内毒素引起炎症反应，其特征是局部产生炎症介质、蛋白质和细胞数量增加。内毒素攻击后2 - 6小时内，支气管肺泡灌洗（BAL）中发现的主要细胞种类从中性粒细胞内流转变为24和48小时内的单核细胞和淋巴细胞内流。在内毒素作用后6小时，BAL促炎活性（即肿瘤坏死因子生物活性和报告细胞上细胞间粘附分子-1的诱导）在内毒素作用后24小时和48小时存在，而不存在。此外，在这种炎症反应中，肺中存在独特的淋巴细胞亚群。

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. Two cohorts are being studied. One cohort of healthy subjects will be studied with bronchoscopy and BAL (up to n = 12) at a single time point. This will provide lung and blood cells to assess immunophenotypes of resident pulmonary cells in the absence of any inflammatory stimulus. The cells will be studied with flow cytometry and with cell culture. Cell-associated and secreted miRNA will be studied ex vivo over a 6-24 hour period. A second cohort of subjects (n = 36) will be studied after undergoing segmental endotoxin challenge with bronchoscopy. BAL will be performed at either 6 (n=12), 24 (n=12) or 48 hours (n=12) after this challenge. Subjects who participated in the first cohort will be eligible to participate in the 2nd cohort with endotoxin challenge after a 4-week interval from their earlier participation. The cells acquired with BAL at these 3 time points will provide a means to study changes in secreted and cell associated miRNA and their relation to specific cell types. To date we have enrolled 25 subjects; 7 completed the control arm of the study (without endotoxin, single bronchoscopy), 14 underwent endotoxin challenge successfully, and 6 subjects either failed screening (4) or withdrew before active participation (2). Preliminary data was accepted for presentation at the American Thoracic Society International Conference (May 2020). The first abstract (Am J of Respir Crit Care Med 2020;201:A7703) described major changes in the transcriptome of the transmigrated neutrophils. Of 19,177 transcripts analyzed with next generation sequencing, 3557 protein coding RNA were upregulated and 2046 were downregulated in BAL compared to blood neutrophils (> 2 fold change, FDR-1%). Interleukin signaling, apoptosis, inflammation mediated by chemokine and cytokine signaling pathway were the top enriched canonical pathways. The significant biological functions were granulocyte chemotaxis, inflammatory response, and defense to bacterium. The molecular and cellular functions associated with these genes that were highly enriched included cellular movement, growth and proliferation, cell-cell signaling, cell death and survival. S. Gairhe , C.Y. Demirkale , S. Alsaaty , D. Reda , P. Torabi-Parizi , A.F. Suffredini. Neutrophil Protein Coding Gene Profiles Detected by RNA Sequencing After Endotoxin-Induced Transmigration to the Lungs in Healthy Volunteers. Am J of Respir Crit Care Med 2020;201:A7703 A second abstract (Am J of Respir Crit Care Med 2020;201:A2971) described the differential expression of non-coding regulatory RNA in transmigrated neutrophils after endotoxin-induced human pulmonary inflammation using nextgeneration sequencing. Non-coding RNA (ncRNA) including microRNA (miRNA), long intergenic noncoding RNA (lincRNA) and antisense RNA are regulators of immune function. The differential expression of the ncRNA was substantially different in the transmigrated neutrophils compared to blood neutrophils. Substantial differential expression of the ncRNA was found (> 2 fold change, 1% FDR, expressed as log2 fold change); miRNA (53 up: 1.2 to 5.4 and 26 down: -1.3 to -5.7), lincRNA (147 up: 1.5 to 7.6 and 64 down: -1.3 to -4.9) and antisense (339 up: 1.1 to 8.2 and 213 down: -1.2 to -5.2). The miRNA were predicted to be involved in the regulation of apoptosis, transcription and cell communication. Only 35% of the upregulated lincRNA and 61% of antisense RNA have attributable functions including roles in cell death, survival, communication and movement. transmigrated human lung neutrophils express regulatory RNA signatures in response to endotoxin that regulate for immune signaling and apoptosis. S. Gairhe , C.Y. Demirkale , S. Alsaaty , D. Reda , P. Torabi-Parizi , A. Suffredini. Differential Expression of Non-Coding Regulatory RNA in Transmigrated Neutrophils During Endotoxin-Induced Human Pulmonary Inflammation Am J of Respir Crit Care Med 2020;201:A2971 https://doi.org/10.1164/ajrccm-conference.2020.201.1_MeetingAbstracts.A2971 S. Gairhe , C.Y. Demirkale , S. Alsaaty , P. Torabi-Parizi , A. Suffredini. Time Dependence of Alveolar Macrophage Transcriptomes After Human Endotoxin Lung Challenge. Am J Respir Crit Care Med 2022;205:A3491 The endotoxin lung challenge model provides a rich opportunity to define molecules and mechanisms involved in the initiation and resolution of pulmonary inflammation. The pandemic curtailed enrollment into this study. Current plans are to complete the study as the pandemic wanes.