Published: 22-02-2021| Version 2 | DOI: 10.17632/vkz52nv6nv.2
Diego Moncada,
Rabindra Tirouvanziam,
Camilla Margaroli


Neutrophils are often considered terminally differentiated and poised for bacterial killing. In chronic diseases such as cystic fibrosis (CF), an unexplained paradox pits massive neutrophil presence against prolonged bacteria infections. Here, we show that neutrophils recruited to CF airways in vivo and in an in vitro transmigration model display rapid and broad transcriptional firing, leading to an upregulation of survival and anabolic genes, and a downregulation of antimicrobial genes. Newly transcribed RNAs are mirrored by the appearance of corresponding proteins, confirming active translation in these cells. Remarkably, treatment by the RNA polymerase II and III inhibitor -amanitin restored expression of key antimicrobial genes, and increased bactericidal capacity of CF airway neutrophils in vitro and in short-term sputum cultures ex vivo. Broadly, our findings show that neutrophil plasticity is regulated at the terminal organ via RNA and protein synthesis, leading to adaptations that profoundly impact their canonical functions (i.e., bacterial clearance).


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Experimental details: Microarray Blood and sputum neutrophils were sorted from patients with CF (N=7) as previously described (Laval et al., 2013). Total RNA was extracted from sorted fractions, mRNA was amplified and cDNA generated (WT-Ovation Pico RNA Amplification System, NuGen). Next, cDNA from sorted neutrophils from sputum and blood sample pairs was labelled with Cy3 and Cy5, respectively, to generate two-color microarray data by competitive hybridization (GE2-v5, Agilent), per manufacturer’s instructions. RNA profiling data were normalized using quantile-normalization with the Robust Multichip Average (RMA) express software v1.1.0 (Bolstad et al., 2003), and analyzed using Gene Set Enrichment Analysis (GSEA, Broad Institute) for significant enrichment of GO terms. Data are shown as expression in airway neutrophils (AW) vs blood (WB). Proteomics Proteins were extracted from purified (blood >98% purity) and transmigrated (LTB4 or CFASN, >98% purity) neutrophils after cell lysis, followed by reduction and alkylation, and digestion with trypsin. The samples were separated into ten fractions using high-pH reversed phase high-performance liquid chromatography (HPLC, pH=10). Each fraction was analyzed by an online liquid chromatography / tandem mass spectrometry (LC-MS/MS, LTQ Orbitrap Elite) system. The spectra were searched against a human proteome database using the SEQUEST algorithm (Eng et al., 1994). Each protein sequence was listed in both forward and reversed orientations to estimate the false discovery rate (FDR) of peptide and protein identifications. The following parameters were used for the search: 10 ppm precursor mass tolerance; 0.5 Da product ion mass tolerance; fully digested with Lys-C; up to two missed cleavages; variable modifications: oxidation of methionine (+15.9949); fixed modifications: carbamidomethylation of cysteine (+57.0214). Peptides with fewer than seven amino acids were deleted, and peptide spectral matches were filtered to <1% FDR. In addition, a protein-level filter was further performed in each data set to reduce the protein-level FDR to be <1%.