Acinetobacter lwoffii exposure promotes PD-L1+ neutrophils and dampens viral-induced type 2 immunity

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.

Methods

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.