Lung neutrophil subsets transcriptomes in WT and CGD mouse in response to fungal PAMPs stimulation

The leukocyte NADPH oxidase 2 (NOX2) regulates inflammation independent of its anti-microbial activity. Inherited defects in NOX2 lead to chronic granulomatous disease (CGD), associated with recurrent bacterial and fungal infections, often with excessive neutrophilic inflammation that results in significant inflammatory burden and tissue damage. We previously showed that excessive leukotriene B4 production by NOX2-deficient mouse neutrophils was a key driver of elevated lung neutrophil infiltration in the initial response to pulmonary challenge with the fungal particle zymosan (Song et al Blood 135, 891-903). We now identify IL-1β and downstream G-CSF as additional amplifying signals that augment and sustain neutrophil accrual in CGD mice. Neutrophils, delivered into the lung via LTB4, were the primary source of IL-1β within the airways, and their increased numbers in CGD lungs led to significantly elevated local and plasma G-CSF. Elevated G-CSF simultaneously promoted increased granulopoiesis and mobilized the release of higher numbers of an immature CD101neg neutrophil subset from the marrow, which trafficked to the lung and acquired a significantly more pro-inflammatory transcriptome in CGD mice compared to WT mice. Thus, neutrophil-produced IL-1β in the acute response to fungal cell walls acts sequentially but non-redundantly with LTB4 to deploy neutrophils and amplify inflammation in CGD mice. This illustrates how NOX2 plays a critical role in dampening multiple components of a feed-forward pipeline for neutrophil recruitment, and highlights NOX2 as a key regulator of neutrophil number and function at inflamed sites. Mice were challenged with or without 20 µg sterile zymosan by intranasal (IN) instillation and euthanized at 24 hours for analysis of inflammation.Total lung was minced and then digested with 5 mg/ml type I collagenase (Worthington) and 1 mg/ml DNAseI (Worthington) in RPMI1640 (Corning) at 37 degree for 45 min and subsequently passed through a 70 μm cell strainer. ACK lysing buffer (Gibco) was used for red blood cell lysis. Lung single-cell suspensions were stained as Live (7-AAD-negative) CD45+CD11b+Ly6G+ along with CD101, and sorted as CD45+CD11b+Ly6G+CD101neg or CD45+CD11b+Ly6G+CD101pos cells on the FACSAria Fusion cell sorter (BD). 50,000-200,000 cells/sample were sorted into RPMI1640 + 20% heat-inactivated FBS, and each group had three replicate samples.