Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis.

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.Keywords: pulmonary macrophages; alveolar macrophages; interstitial macrophages; macrophage polarization; lung fibrosis Cells of the innate immune system, and especially myeloid cells such as neutrophils, eosinophils, monocytes, macrophages (alveolar and interstitial), and dendritic cells (DCs, i.e., plasmacytoid DCs, CD1031 DCs, and CD11b 1 DCs), play an important role in lung development and physiology, and contribute to important lung diseases, including pulmonary infection, cancer, asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis (1-5). Alveolar and interstitial lung macrophages exhibit different origins and life spans in lungs, and have been identified as key regulators of pathological and reparative processes. Alveolar macrophages, which are considered tissue-resident macrophages, populate lung tissue during early embryogenesis and remain viable for prolonged periods, with minimal replenishment from bone marrow-derived monocytes (6). In contrast, interstitial macrophages originate from bone marrow-derived monocytes and have a shorter half-life (7,8). In recent studies, several groups of investigators suggested that these two populations of lung macrophages play opposing roles in lung injury. Alveolar macrophages appear to limit neutrophil influx into the lung during acute lung injury (9) or chronic exposure to organic dust (10), whereas interstitial macrophages promote neutrophil extravasation (11,12). An additional layer of complexity is added by the phenotypic plasticity of macrophages. Classically activated macrophages (sometimes referred to as M1-polarized) have been suggested to promote the development of acute lung injury, whereas alternatively activated macrophages (M2) may play a role in limiting or resolving lu...

Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) 1 . 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 other types of pneumonia 2 . 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 subjected them to flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA-seq on 10 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours 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-gamma 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 harboring SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

The factors responsible for maintaining persistent organ fibrosis in systemic sclerosis (SSc) are not known but emerging evidence implicates toll-like receptors (TLRs) in the pathogenesis of SSc. Here we show the expression, mechanism of action and pathogenic role of endogenous TLR activators in skin from patients with SSc, skin fibroblasts, and in mouse models of organ fibrosis. Levels of tenascin-C are elevated in SSc skin biopsy samples, and serum and SSc fibroblasts, and in fibrotic skin tissues from mice. Exogenous tenascin-C stimulates collagen gene expression and myofibroblast transformation via TLR4 signalling. Mice lacking tenascin-C show attenuation of skin and lung fibrosis, and accelerated fibrosis resolution. These results identify tenascin-C as an endogenous danger signal that is upregulated in SSc and drives TLR4-dependent fibroblast activation, and by its persistence impedes fibrosis resolution. Disrupting this fibrosis amplification loop might be a viable strategy for the treatment of SSc.

Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodeling, and fibrosis) are attenuated in the lungs of Vim-/- mice challenged with LPS, bleomycin, and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim-/- and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests vimentin may be a key regulator of the NLRP3 inflammasome.