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RNA sequencing analysis of unchallenged, PBS injected and S. pneumoniae infected pcsk9 KO and WT zebrafish

ABSTRACT: Purpose: S. pneumoniae infection in both zebrafish larvae and adult fish can be used to model pneumococcal bacteremia. Here, we used unchallenged, PBS injected and S. pneumoniae (T4) infected adult zebrafish to study the genome-wide role of pcsk9 in vivo in steady-state and upon immune system stimulation. Methods: Adult pcsk9 KO zebrafish and their WT siblings were infected with S. pneumoniae (3,370,000 CFU; SD 840,000 CFU) and their total liver RNA isolated at 1-day post infection. RNA-sequencing data was produced by NovaSeq. Data processing pipeline was built on Snakemake wrappers, and used STAR to quantify features from quality and adapter trimmed reads in gene level. Prior to the normalisation and differential gene expression analysis with DESeq2, the matrix of raw gene counts was prefiltered to keep only rows that have at least 10 reads total and then split by treatment to analyze each case separately. Result tables were annotated with biomaRt. Results: Snakemake preprocessing pipeline measured 32,520 genes against GRCz11, ENSEMBL release 97. 20,709 of them was used for statistical testing. Full result tables for each treatment were ranked by p-value and the normalized DESeq2 abundances in samples were included. The genes with BH-adjusted p-value <0.05 was considered differentially expressed. Conclusions: Our study identified up- and down-regulated genes in unchallenged, PBS injected and pneumococcal infected pcsk9 KO zebrafish liver compared to WT zebrafish.

ORGANISM(S): Danio rerio

PROVIDER: GSE165508 | GEO | 2021/07/02

REPOSITORIES: GEO

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Project description:Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes. Here, we demonstrate that the viral hemagglutinin (HA) mediates bacterial OM by inducing a pro-inflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings show that it is the inflammatory response that mediates pneumococcal replication; not viral suppression of the immune system or epithelial damage. This study provide the first evidence that HA induced inflammation drives pneumococcal replication in the middle ear cavity, which has important consequences to the treatment of pneumococcal OM. Five-day old C57BL/6 mice were colonised intranasally (i.n.) with 2M-CM-^W103 colony forming units (CFU) of S. pneumoniae EF3030Lux in 3 M-BM-5Ls of PBS. Alternatively, mice were mock-infected with an equivalent volume of phosphate buffered saline (PBS). At 14-days of age, infant mice were infected i.n. with 20 plaque forming units (PFU) (PR8/34, Cambridge/34 and WSN/33) or 102.5 PFU (all other virus strains) of egg-grown IAV in 3 M-BM-5Ls of PBS. Viral doses were selected to ensure a reproducible infection with minimum morbidity and no mortality. Six days post-IAV infection, mice were euthanised and organs were collected for analysis. Six independent biological replicates (where both ears from one mouse were pooled to create one sample) were used for each condition and analyzed using the NimbleGen platform (12M-CM-^W135K Mouse Gene Expression Arrays, Roche Nimblegen, USA). Indirect labeling, hybridization and washing was performed according to the manufacturerM-bM-^@M-^Ys instructions. Array images were acquired with a NimbleGen MS200 scanner, and images were processed with NimbleScan software using the RMA algorithm. Data was processed using Arraystar (DNASTAR, USA) with default settings as described in the manual. Differential expression tests were performed with a moderated T-test implemented in Arraystar, followed by FDR correction of the P values (Q-values) according to the method of Storey and Tibshirani

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RNA sequencing analysis of unchallenged, PBS injected and S. pneumoniae infected pcsk9 KO and WT zebrafish

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