Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined.Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes Liver X Receptor (LXR)-dependent clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h -/mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h and nonhematopoietic Ch25h deletion is sufficient to confer lung protection. In acute respiratory distress syndrome patients, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting upon different cell types in the lung communicates distinct, temporal LXRindependent and -dependent signals to regulate inflammatory homeostasis.

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.

Importance: Tick–borne diseases (TBD) including Spotted Fever Group Rickettsiosis (SFGR), ehrlichiosis, and increasingly Lyme disease represent a substantial public health concern throughout much of the Southeastern United States. Yet, there is uncertainty about the epidemiology of these diseases due to pitfalls in existing diagnostic test methodologies. Objective: To examine patterns of diagnostic testing and incidence of TBD in a large, academic healthcare system. Design: Cross–sectional study of diagnostic test results from UNC Health for the period January 1st, 2017 to November 30th, 2020. Setting: Large, academic healthcare system in central North Carolina including inpatient and outpatient facilities. Participants: All Individuals seeking routine care at UNC Health facilities who had testing for SFGR, ehrlichiosis or Lyme disease performed during the study period Measurements: Rates of test positivity, testing completeness, and incidence of TBD Results: Among the 20,528 diagnostic tests performed, we identified 47 laboratory–confirmed, incident cases of SFGR, 27 of ehrlichiosis, and 76 of Lyme, representing incidence rates of 4.7%, 7.1%, and 0.7% respectively. However, 79.3% of SFGR tests and 74.3% Ehrlichia tests lacked a paired convalescent sample. The total number of tests for Lyme disease was more than SFGR and ehrlichiosis combined, despite the relatively low incidence of disease in region. Most striking, testing for ehrlichiosis was performed in only half of patients in whom SFGR was ordered, suggesting that this disease remains underrecognized. Overall, we estimate that there were 187 incident cases of SFGR and 309 of ehrlichiosis that were not identified due to incomplete testing; a number that would drastically increase – and in the case of ehrlichiosis, nearly double – the total number of cases reported. Conclusions and Relevance: A majority of patients suspected of having TBD did not have testing performed in accordance with established guidelines, substantially limiting our understanding of TBD epidemiology. Furthermore, there appears to be a large discrepancy between the local burden of disease and the testing that is performed. These findings underscore the need to pursue more robust, active surveillance strategies to estimate the burden of TBDs and distribution of causative pathogens.