Single-base editing of rs12603332 on chromosome 17q21 with a cytosine base editor regulates ORMDL3 and ATF6α expression.

BACKGROUND: Genetic association studies have demonstrated that the SNP rs12603332 located on chromosome 17q21 is highly associated with the risk of the development of asthma. METHODS: To determine whether SNP rs1260332 is functional in regulating levels of ORMDL3 expression, we used a Cytosine Base Editor (CBE) plasmid DNA or a CBE mRNA to edit the rs12603332 C risk allele to the T non-risk allele in a human lymphocyte cell line (i.e., Jurkat cells) and in primary human CD4 T cells that carry the C risk alleles. RESULTS: Jurkat cells with the rs12603332 C risk allele expressed significantly higher levels of ORMDL3 mRNA, as well as the ORMDL3 regulated gene ATF6α as assessed by qPCR compared to Jurkat clones with the T non-risk allele. In primary human CD4 T cells, we edited 90 ± 3% of the rs12603332-C risk allele to the T non-risk allele and observed a reduction in ORMDL3 and ATF6α expression. Bioinformatic analysis predicted that the non-risk allele rs12603332-T could be the central element of the E-box binding motif (CANNTG) recognized by the E47 transcription factor. An EMSA assay confirmed the bioinformatics prediction demonstrating that a rs12603332-T containing probe bound to the transcription factor E47 in vitro. CONCLUSIONS: SNP rs12603332 is functional in regulating the expression of ORMDL3 as well as ORMDL3 regulated gene ATF6α expression. In addition, we demonstrate the use of CBE technology in functionally interrogating asthma-associated SNPs using studies of primary human CD4 cells.

Differential lung inflammation and injury with tobacco smoke exposure in Wistar Kyoto and spontaneously hypertensive rats.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and has been associated with periods of intense lung inflammation. The objective of this study was to characterize whether similar rat strains, possessing different genetic predispositions, might play a role in exacerbating the pathophysiology of COPD-like cellular and structural changes with progressive 12-week exposure to tobacco smoke (TS). Normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats were compared. MATERIALS AND METHODS: WKY and SH rats were exposed to filtered air or to tobacco smoke at a particulate concentration of 80 mg/m3 for 4, 8, or 12 weeks. Necropsy was performed 24 h after the last exposure to obtain cells by bronchoalveolar lavage for total cell and differential counts. Scoring of lung tissues and immunohistochemical staining for M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages were performed on paraffin-embedded lung sections. RESULTS AND DISCUSSION: With progressive exposure, TS-exposed SH rats demonstrated significant airspace enlargement, mucin production, and lung inflammation compared to their FA control and TS-matched WKY rats. Moreover, SH rats also demonstrated increased expression of the M1 marker in alveolar macrophages compared to FA control, as well as the M2 marker compared to controls and TS-exposed WKY rats. CONCLUSION: The progressive tobacco smoke exposure contributes to persistent lung injury and inflammation that can be significantly enhanced by rat strain susceptibility in the genesis of COPD.

Sex and strain-based inflammatory response to repeated tobacco smoke exposure in spontaneously hypertensive and Wistar Kyoto rats.

CONTEXT: Approximately four million people die every year from chronic obstructive pulmonary disease (COPD), with more than 80% of the cases attributed to smoking. OBJECT: The purpose of this study was to examine the rat strain and sex-related differences and the extended tobacco smoke exposure to induce lung injury and inflammation with the goal of finding a suitable rodent model to study COPD. METHODS: Male and female spontaneously hypertensive (SH) and male Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or to tobacco smoke (TS: 90 mg/m3 particulate concentration) for 6 h/day, three days/week for 4 or 12 weeks. RESULTS: Male SH rats demonstrated an enhanced, persistent inflammatory response compared to female SH and male WKY rats with extended TS exposure. Following four weeks of TS exposure, male SH rats had significantly increased total leukocytes and macrophage numbers, levels of TNF-alpha and elevated lactate dehydrogenase activity in bronchoalveolar lavage fluid compared with female SH, male WKY rats and corresponding controls. After 12 weeks of TS exposure, male SH rats continued to show significant increase in inflammatory cells and TNF-alpha, as well as IL-6 mRNA lung expression. In addition, the alveolar airspace of male SH rats exposed to TS was significantly enlarged compared to their FA controls, female SH and WKY rats. CONCLUSION: The male SH rat demonstrates greater cellular, inflammatory and structural changes highly reminiscent of COPD compared to female SH and male WKY rats, suggesting that the male SH rat is an optimal rodent model to study COPD.

ORMDL3 expression in ASM regulates hypertrophy, hyperplasia via TPM1 and TPM4, and contractility.

ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function, we used Cre-loxP techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre), which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), developed hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3-mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro, which was associated with increased levels of intracellular Ca2+; increased cell surface membrane Orai1 Ca2+ channels, which mediate influx of Ca2+ into the cytoplasm; and increased expression of ASM contractile genes sarco/endoplasmic reticulum Ca2+ ATPase 2b and smooth muscle 22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and airway hyperreactivity (AHR). ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility), which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma, which is highly linked to ORMDL3 on chromosome 17q12-21.

Differential pulmonary effects of wintertime California and China particulate matter in healthy young mice.

Airborne particulate matter (PM) is associated with adverse cardiorespiratory effects. To better understand source-orientated PM toxicity, a comparative study of the biological effects of fine PM (diameter≤2.5µm, PM2.5) collected during the winter season from Shanxi Province, China, and the Central Valley, California, United States, was conducted. The overarching hypothesis for this study was to test whether the chemical composition of PM on an equal mass basis from two urban areas, one in China and one in California, can lead to significantly different effects of acute toxicity and inflammation in the lungs of healthy young mice. Male, 8-week old BALB/C mice received a single 50µg dose of vehicle, Taiyuan PM or Sacramento PM by oropharyngeal aspiration and were sacrificed 24h later. Bronchoalveolar lavage, ELISA and histopathology were performed along with chemical analysis of PM composition. Sacramento PM had a greater proportion of oxidized organic material, significantly increased neutrophil numbers and elevated CXCL-1 and TNF-α protein levels compared to the Taiyuan PM. The findings suggest that Sacramento PM2.5 was associated with a greater inflammatory response compared to that of Taiyuan PM2.5 that may be due to a higher oxidice. Male, 8-week old BALB/C mice received a single 50µg dose of vehicle, Taiyuan PM or Sacramento PM by oropharyngeal aspiration and were sacrificed 24h later. Bronchoalveolar lavage, ELISA and histopathology were performed along with chemical analysis of PM composition. Sacramento PM had a greater proportion of oxidized organic material, significantly increased neutrophil numbers and elevated CXCL-1 and TNF-α protein levels compared to the Taiyuan PM. The findings suggest that Sacramento PM2.5 was associated with a greater inflammatory response compared to that of Taiyuan PM2.5 that may be due to a higher oxidized state of organic carbon and copper content.