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The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.

Augmented glycolysis due to metabolic reprogramming in lung myofibroblasts is critical to their profibrotic phenotype. The primary glycolysis byproduct, lactate, is also secreted into the extracellular milieu, together with which myofibroblasts and macrophages form a spatially restricted site usually described as fibrotic niche. Therefore, we hypothesized that myofibroblast glycolysis might have a non-cell autonomous effect through lactate regulating the pathogenic phenotype of alveolar macrophages. Here, we demonstrated that there was a markedly increased lactate in the conditioned media of TGF-β1 (transforming growth factor-β1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-β1- or bleomycin-induced lung fibrosis. Importantly, the media and BALFs promoted profibrotic mediator expression in macrophages. Mechanistically, lactate induced histone lactylation in the promoters of the profibrotic genes in macrophages, consistent with the upregulation of this epigenetic modification in these cells in the fibrotic lungs. The lactate inductions of the histone lactylation and profibrotic gene expression were mediated by p300, as evidenced by their diminished concentrations in p300-knockdown macrophages. Collectively, our study establishes that in addition to protein, lipid, and nucleic acid molecules, a metabolite can also mediate intercellular regulations in the setting of lung fibrosis. Our findings shed new light on the mechanism underlying the key contribution of myofibroblast glycolysis to the pathogenesis of lung fibrosis.

Sepsis and trauma cause inflammation and elevated susceptibility to hospital-acquired pneumonia. As phagocytosis by macrophages plays a critical role in the control of bacteria, we investigated the phagocytic activity of macrophages after resolution of inflammation. After resolution of primary pneumonia, murine alveolar macrophages (AMs) exhibited poor phagocytic capacity for several weeks. These paralyzed AMs developed from resident AMs that underwent an epigenetic program of tolerogenic training. Such adaptation was not induced by direct encounter of the pathogen but by secondary immunosuppressive signals established locally upon resolution of primary infection. Signal-regulatory protein α (SIRPα) played a critical role in the establishment of the microenvironment that induced tolerogenic training. In humans with systemic inflammation, AMs and also circulating monocytes still displayed alterations consistent with reprogramming six months after resolution of inflammation. Antibody blockade of SIRPα restored phagocytosis in monocytes of critically ill patients in vitro, which suggests a potential strategy to prevent hospital-acquired pneumonia. Sepsis and physical trauma can increase the susceptibility of patients to pneumonia. Roquilly and colleagues demonstrate that sepsis results in durable impairment of alveolar phagocytic function that is dependent on the localized expression of the inhibitory receptor SIRPα.

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome 1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia 2 . Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation. Analysis of bronchoalveolar lavage fluid samples from patients with SARS-CoV-2-induced respiratory failure suggests that SARS-CoV-2 infects alveolar macrophages to cause release of T cell chemoattractants, thereby inducing local inflammatory cytokine release and further T cell activation, ultimately resulting in a positive feedback loop that drives alveolar inflammation.

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1 + SiglecF + transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury. Using scRNA-seq analysis, Bhattacharya and colleagues identify a subset of profibrotic lung macrophages that have a gene expression signature intermediate between those of monocytes and alveolar macrophages.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation. Control of macrophage activation in the lungs is essential for the prevention of tissue damage. MacDonald and colleagues show that alveolar macrophages have impaired glycolysis and are hyporesponsive during type 2 inflammation in a manner controlled by the lung environment.

Macrophages have long been considered as particularly plastic cells. However, recent work combining fate mapping, single-cell transcriptomics and epigenetics has undermined the macrophage plasticity dogma. Here, we discuss recent studies that have carefully dissected the response of individual macrophage subsets to pulmonary insults and call for an adjustment of the macrophage plasticity concept. We hypothesize that prolonged tissue residency shuts down much of the plasticity of macrophages and propose that the restricted plasticity of resident macrophages has been favored by evolution to safeguard tissue homeostasis. Recruited monocytes are more plastic and their differentiation into resident macrophages during inflammation can result in a dual imprinting from both the ongoing inflammation and the macrophage niche. This results in inflammation-imprinted resident macrophages, and we speculate that rewired niche circuits could maintain this inflammatory state. We believe that this revisited plasticity model offers opportunities to reset the macrophage pool after a severe inflammatory episode. Based on the results of recent studies that have dissected the response of individual macrophage subsets to pulmonary insults, Guilliams and Svedberg call for an adjustment of the macrophage plasticity concept.

Acute lung injury (ALI) and its severe form, known as acute respiratory distress syndrome (ARDS), are caused by direct pulmonary insults and indirect systemic inflammatory responses that result from conditions such as sepsis, trauma, and major surgery. The reciprocal influences between pulmonary and systemic inflammation augments the inflammatory process in the lung and promotes the development of ALI. Emerging evidence has revealed that alveolar macrophage (AM) death plays important roles in the progression of lung inflammation through its influence on other immune cell populations in the lung. Cell death and tissue inflammation form a positive feedback cycle, ultimately leading to exaggerated inflammation and development of disease. Pharmacological manipulation of AM death signals may serve as a logical therapeutic strategy for ALI/ARDS. This review will focus on recent advances in the regulation and underlying mechanisms of AM death as well as the influence of AM death on the development of ALI.

Loke and colleagues show that vitamin A is required for the conversion of interleukin 4 (IL-4)-activated monocyte-derived macrophages into macrophages with a tissue-resident phenotype in the peritoneal cavity and in S. mansoni –induced liver granulomas in mice. It remains unclear whether activated inflammatory macrophages can adopt features of tissue-resident macrophages, or what mechanisms might mediate such a phenotypic conversion. Here we show that vitamin A is required for the phenotypic conversion of interleukin 4 (IL-4)-activated monocyte-derived F4/80 int CD206 + PD-L2 + MHCII + macrophages into macrophages with a tissue-resident F4/80 hi CD206 − PD-L2 − MHCII − UCP1 + phenotype in the peritoneal cavity of mice and during the formation of liver granulomas in mice infected with Schistosoma mansoni . The phenotypic conversion of F4/80 int CD206 + macrophages into F4/80 hi CD206 − macrophages was associated with almost complete remodeling of the chromatin landscape, as well as alteration of the transcriptional profiles. Vitamin A–deficient mice infected with S. mansoni had disrupted liver granuloma architecture and increased mortality, which indicates that failure to convert macrophages from the F4/80 int CD206 + phenotype to F4/80 hi CD206 − may lead to dysregulated inflammation during helminth infection.