A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.

BACKGROUND: Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer's disease (AD), possibly through limiting neuroinflammatory responses. METHODS: To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. RESULTS: Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. CONCLUSIONS: Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose.

The CLIP-CHIP oligonucleotide microarray: dedicated array for analysis of all protease, nonproteolytic homolog, and inhibitor gene transcripts in human and mouse.

The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples. After hybridization, the fluorescence intensity of each spot is measured, thus identifying mRNA transcripts that are expressed and allowing basic quantification of expressed transcripts.

BBGD: an online database for blueberry genomic data.

BACKGROUND: Blueberry is a member of the Ericaceae family, which also includes closely related cranberry and more distantly related rhododendron, azalea, and mountain laurel. Blueberry is a major berry crop in the United States, and one that has great nutritional and economical value. Extreme low temperatures, however, reduce crop yield and cause major losses to US farmers. A better understanding of the genes and biochemical pathways that are up- or down-regulated during cold acclimation is needed to produce blueberry cultivars with enhanced cold hardiness. To that end, the blueberry genomics database (BBDG) was developed. Along with the analysis tools and web-based query interfaces, the database serves both the broader Ericaceae research community and the blueberry research community specifically by making available ESTs and gene expression data in searchable formats and in elucidating the underlying mechanisms of cold acclimation and freeze tolerance in blueberry. DESCRIPTION: BBGD is the world's first database for blueberry genomics. BBGD is both a sequence and gene expression database. It stores both EST and microarray data and allows scientists to correlate expression profiles with gene function. BBGD is a public online database. Presently, the main focus of the database is the identification of genes in blueberry that are significantly induced or suppressed after low temperature exposure. CONCLUSION: By using the database, researchers have developed EST-based markers for mapping and have identified a number of "candidate" cold tolerance genes that are highly expressed in blueberry flower buds after exposure to low temperatures.

The CLIP-CHIP oligonucleotide microarray: dedicated array for analysis of all protease, nonproteolytic homolog, and inhibitor gene transcripts in human and mouse.

The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples. After hybridization, the fluorescence intensity of each spot is measured, thus identifying mRNA transcripts that are expressed and allowing basic quantification of expressed transcripts.