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Transcriptome sequencing analysis of naïve CD4+ T cells treated with NETs or control vehicle

ABSTRACT: Purpose: We found neutrophil extracellular traps (NETs) can affect the differentiation of naïve CD4+ T cells. This sequencing aims to explore the gene profile change of neutrophil extracellular traps (NETs) on naïve CD4+ T cells. Methods: Total RNA was extracted from the T cells treated with NETs or vehicle for 72h and send to sequencing. Sequencing libraries were generated using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) and sequenced on an Illumina platform. Paired-end reads were then generated and evaluated for quality control. Clean raw data with high quality were aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. For quantification, FeatureCouts was used to count the read numbers mapped of each gene. Differential expression analysis between two groups (three biological replicates per condition) was performed using DESeq2 R package. Results: Using an optimized data analysis workflow, we mapped around 40 million sequence reads per sample with total mapping rate around 96%. Total 23,249 transcripts in the NETs and Vehicle group were identified. Approximately 7% of the transcripts (1675) showed differential expression between the with p value <0.05. Top differential expressed genes were then enriched based on KEGG and GO database. Altered expression genes in the most enrichment pathways were confirmed with qRT–PCR and western blot, demonstrating the high degree of sensitivity of the RNA-seq method. The top differential expressed genes provide new insigts for the mechanism of T cell differentiation affected by NETs. Conclusions: Our study represents the first detailed analysis of naïve CD4+ T cells treated with NETs. Most differentital expressed genes were enriched in metabolic pahtways, which prompted us to further explore the related mechanism of the novel phenotype.

ORGANISM(S): Mus musculus

PROVIDER: GSE161464 | GEO | 2021/12/10

REPOSITORIES: GEO

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Project description:Introduction: Autoreactivity to histones is a pervasive feature of several human autoimmune disorders including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. Methods: We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. Results: We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the IgG and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Conclusions: Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified. Serum samples from 20 systemic lupus erythematosis patients were run on the Human Epigenome Microarray Platform V1.0 (HEMP; a single-color platform), in order to profile their autoantibodies against a library of post-translationally modified histone peptides. These 20 samples were randomly selected from a larger cohort previously profiled (data not shown) on the Utz Lab Whole Protein Autoantigen Array V2.0 (a single-color platform), where 14 were histone-reactive and 6 were histone-nonreactive. Control sera from 9 healthy adults and a positive control comprising a mixture of autoimmune sera with defined reactivities, were also run on HEMP V1.0. Together, these samples comprise the data appearing in Figures 1 and S1 (IgG and IgM isotype reactivity profiles, respectively), identifying IgG reactivity to 9 peptides that significantly distinguish histone-reactive from -nonreactive sera among 96 peptides profiled. For data appearing in Figure 5, serum samples from a total of 6 Balb/c mice, consisting of two treatment groups, NETs (Neutrophil Extracellular Traps) and NETs + CRAMP (cathelicidin-related antimicrobial peptide) were collected monthly over a 3-month period, along with a zero time point. These samples were compared with a positive control consisting of serum collected from a MLR/lpr mice exhibiting lupus-like symptoms, and a negative control with no serum. The 0, 1 and 2 month time points were profiled on the Utz Lab Whole Protein Autoantigen Array V2.0 and are shown in Figure 5A-B, while the 1 and 3 month time points were profiled on HEMP V1.0 arrays and shown in Figure 5E. All samples were run once with no replicates.

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Transcriptome sequencing analysis of naïve CD4+ T cells treated with NETs or control vehicle

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