Respiratory syncytial virus (RSV) infection in infants and toddlers is a major public health problem. Here, we provide a protocol for neonatal RSV infection in mice and immune analysis of infected lungs and bronchoalveolar lavage (BAL) fluid. We describe steps for anesthesia and intranasal inoculation, weight monitoring, and whole lung collection. We then detail BAL fluid immune and whole lung analyses. This protocol can be used for neonatal pulmonary infection with other viruses or bacteria.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

In severe bacterial infections, there is a pro-inflammatory response to promote bacterial clearance but this response can cause tissue injury. Later, the immune system becomes dysregulated and the host is unable to clear a secondary or a pre-existing infection. Specialized Pro-resolving Mediators (SPMs) such as resolvin D2 (RvD2) have been shown to be beneficial for inflammation/infection resolution in animal models of sepsis but in vivo mechanisms by which RvD2 may promote bacterial clearance and/or attenuate deleterious effects of a secondary infection have not been fully established. In this study, we used the 2-hit model of cecal ligation and puncture (CLP) induced infectious peritonitis and secondary lung infection with Pseudomonas aeruginosa to find possible antimicrobial and immunomodulatory mechanisms of RvD2. We show that RvD2 given as late as 48h after CLP surgery reduced blood bacterial load without altering plasma cytokines compared to mice given saline vehicle. RvD2 increased splenic neutrophil accumulation as well as average reactive oxygen species (ROS) production. There was also an increase in an immature leukocyte population the myeloid derived suppressor cells (MDSCs) in the spleen of RvD2 treated mice. RvD2 reduced lung lavage bacterial load 24h after P. aeruginosa administration and significantly decreased lung lavage levels of IL-23, a cytokine essential in the Th-17 inflammatory response. In addition, we show that RvD2 increased the number of non-inflammatory alveolar macrophages after P. aeruginosa administration compared to saline treated mice. The study uncovered an antimicrobial mechanism of RvD2 where RvD2 increases mature neutrophil and MDSC accumulation into the spleen to promote blood bacterial clearance. The study showed that in this 2-hit model, RvD2 promotes lung bacterial clearance, increased non-inflammatory alveolar macrophage number and inhibits an adaptive immune pathway providing evidence of its resolution mechanism in secondary pulmonary infection.
Copyright © 2022 Sundarasivarao, Walker, Rodriguez, Spur and Yin.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor β (TGF-β) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-β signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.
© 2022. The Author(s).

Chronic exposure to airborne carbon black ultrafine (nCB) particles generated from incomplete combustion of organic matter drives IL-17A-dependent emphysema. However, whether and how they alter the immune responses to lung cancer remains unknown. Here, we show that exposure to nCB particles increased PD-L1+ PD-L2+ CD206+ antigen-presenting cells (APCs), exhausted T cells, and Treg cells. Lung macrophages that harbored nCB particles showed selective mitochondrial structure damage and decreased oxidative respiration. Lung macrophages sustained the HIF1α axis that increased glycolysis and lactate production, culminating in an immunosuppressive microenvironment in multiple mouse models of non-small cell lung cancers. Adoptive transfer of lung APCs from nCB-exposed wild type to susceptible mice increased tumor incidence and caused early metastasis. Our findings show that nCB exposure metabolically rewires lung macrophages to promote immunosuppression and accelerates the development of lung cancer.