DOI: 10.5061/dryad.r7sqv9snn
title: Acinetobacter lwoffii exposure promotes PD-L1+ neutrophils and dampens viral-induced type 2 immunity

description: Severe, recurrent viral infections in early life, such as those caused by
 respiratory syncytial virus (RSV) are associated with wheezing and asthma.
 Exposure to farmyard microbes protects against allergen-induced asthma,
 but their role in viral-induced airways disease is less understood. Here
 neonatal murine recurrent RSV infection promoted pulmonary eosinophilia,
 mucus hypersecretion and IL-13+ CD4+ T cells. These pathological features
 were ameliorated by concomitant inhalation of inactivated cowshed-derived
 bacteria, Acinetobacter lwoffii F78. The protective effect was maximal
 with continuous A.lwoffii exposure and protection correlated with
 increased circulatory, airway and lung parenchymal neutrophils. However,
 in contrast to pro-inflammatory neutrophils induced by RSV infection,
 transcriptomic and phenotypic analysis revealed A.lwoffii induced a
 distinct neutrophil population with reduced expression of CXCR4 and CD101,
 but elevated PD-L1. Induction of these PD-L1+ immunoregulatory neutrophils
 by inhaled A.lwoffii was linked to a concomitant reduction in Th2
 immunity.
description: To obtain extravascular and intravascular pulmonary
 neutrophils for bulk RNA sequencing, neonatal mice received labelling
 antibody PE anti-mouse Ly-6G antibody (clone 1A8, 1µg of antibody in 100
 µl of sterile PBS per mouse) (Biolegend) intravenously 3 minutes before
 euthanisation. Lung tissues were harvested without performing
 bronchoalveolar lavage. Neutrophil enrichment was performed from recovered
 lung cells using Neutrophil Isolation Kit (Miltenyi Biotec, location) via
 magnetic negative selection. Cells were then stained with DAPI and BV605
 anti-mouse Ly-6G antibody (clone 1A8, 1: 200 dilution) (Biolegend) in FACS
 sorting buffer (PBS, 25mM HEPES, 1mM EDTA 2% FCS, 0.2mm sterile filtered)
 for 20 minutes at 4oC, protected from light. Intravascular neutrophils
 (DAPI+ and PE Ly-6G+) and extravascular neutrophils (DAPI+, BV605 Ly-6G+
 and PE Ly-6G-) were sorted using BD FACS Melody with a nozzle size of 100
 microns. Sorted neutrophils were lysed in RLT lysis buffer with the
 addition of b-Mercaptoethanol for storage.   
 RNA extraction was performed using TRIzolTM (Thermo
 Fisher) and Isopropanol. RNA quantity and sample quality (RIN) were
 determined using TapeStation (Agilent, location). Due to low RNA yield,
 samples were pooled from three biological samples within the same group
 and sequenced by Genewiz (AZENTA, US) using Illumina ® NovaSeqTM or HiSeq®
 platforms.
description: # Acinetobacter lwoffii exposure promotes PD-L1+ neutrophils and dampens
 viral-induced type 2 immunity
 [https://doi.org/10.5061/dryad.r7sqv9snn](https://doi.org/10.5061/dryad.r7sqv9snn) ## Description of the data and file structure ### Files and variables #### File: R2I\_R2\_001.fastq.gz.md5 \**Description: ** Intravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: R2I\_R1\_001.fastq.gz.md5 **Description:** Intravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: R2E\_R2\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: R2I\_R2\_001.fastq.gz **Description:** Intravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: R2E\_R1\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: R2I\_R1\_001.fastq.gz **Description:** Intravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: PBSI\_R2\_001.fastq.gz.md5 **Description:** Intravascular pulmonary neutrophil from PBS treated mice_sample 1 #### File: R2E\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: PBSI\_R1\_001.fastq.gz.md5 **Description:** Intravascular pulmonary neutrophil from PBS treated mice_sample 1 #### File: R2E\_R1\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from ReRSV infected mice_sample 2 #### File: AR2E\_R2\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 2 #### File: PBSI\_R2\_001.fastq.gz **Description:** Intravascular pulmonary neutrophil from PBS treated mice_sample 1 #### File: AR2E\_R1\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 2 #### File: PBSI\_R1\_001.fastq.gz **Description:** Intravascular pulmonary neutrophil from PBS treated mice_sample 1 #### File: AR1I\_R2\_001.fastq.gz.md5 **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: AR2E\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 2 #### File: AR1I\_R1\_001.fastq.gz.md5 **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: AR2E\_R1\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 2 #### File: AR1E\_R2\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: AR1I\_R2\_001.fastq.gz **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: AR1E\_R1\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: AR1I\_R1\_001.fastq.gz **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: A2E\_R2\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 2 #### File: AR1E\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: A2E\_R1\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 2 #### File: AR1E\_R1\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated ReRSV infected mice_sample 1 #### File: A1l\_R2\_001.fastq.gz.md5 **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A2E\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 2 #### File: A1l\_R1\_001.fastq.gz.md5 **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A2E\_R1\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 2 #### File: A1E\_R2\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A1l\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A1E\_R1\_001.fastq.gz.md5 **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A1l\_R1\_001.fastq.gz **Description:** Intratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A1E\_R2\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 #### File: A1E\_R1\_001.fastq.gz **Description:** Extratravascular pulmonary neutrophil from A.lwoffii treated mice_sample 1 ## Code/software Bioinformatics services were provided by Genewiz and analysis package included FastQC quality assessment, trimming, sequence alignment, count generation and differential gene expression analysis. Independent transcriptomic analysis was performed using various free, open and publicly available online flatforms. Galaxy Europe was used to perform initial sequence quality assessment on paired FASTQ files from all samples provided by Genewiz. Briefly, sequence quality was assessed by fastQC (Version 0.11.9). Technical sequences such as adaptors that were artificially introduced into the sequence during library preparation (Nextera, Illumina) were trimmed out using Cutadapt (Version 4.0) . Alignment to mouse reference genome GRCm39 (Genome Reference Consortium Mouse Build 39) were performed using STAR (Version 2.7.8a) (*59*). Hit counts to known genes were calculated by FeatureCounts. Count matrix were generated by combining hit counts of all samples and used for downstream differential gene expression analysis. Differential gene expression analysis between experimental and control groups was also performed on Galaxy Europe using DESeq2. K-means clustering analysis was performed on an open web application iDEP (1) . Enriched biological pathways were identified using Gene Set Enrichment Analysis (GSEA). (1) S. X. Ge, E. W. Son, R. Yao, iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. *BMC Bioinformatics* **19**, 534 (2018)