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Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-F resident alveolar MΦ and a mixed population of CD11b MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP) to CD11b MΦ. Upon loss of c-FLIP, CD11b MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11b MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11b MΦ, but not Siglec-F MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11b MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.

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OBJECTIVES: Prone position ventilation is a potentially life-saving ancillary intervention but is not widely adopted for coronavirus disease 2019 or acute respiratory distress syndrome from other causes. Implementation of lung-protective ventilation including prone positioning for coronavirus disease 2019 acute respiratory distress syndrome is limited by isolation precautions and personal protective equipment scarcity. We sought to determine the safety and associated clinical outcomes for coronavirus disease 2019 acute respiratory distress syndrome treated with prolonged prone position ventilation without daily repositioning. DESIGN: Retrospective single-center study. SETTING: Community academic medical ICU. PATIENTS: Sequential mechanically ventilated patients with coronavirus disease 2019 acute respiratory distress syndrome. INTERVENTIONS: Lung-protective ventilation and prolonged protocolized prone position ventilation without daily supine repositioning. Supine repositioning was performed only when Fio 2 less than 60% with positive end-expiratory pressure less than 10 cm H2O for greater than or equal to 4 hours. MEASUREMENTS AND MAIN RESULTS: Primary safety outcome: proportion with pressure wounds by Grades (0–4). Secondary outcomes: hospital survival, length of stay, rates of facial and limb edema, hospital-acquired infections, device displacement, and measures of lung mechanics and oxygenation. Eighty-seven coronavirus disease 2019 patients were mechanically ventilated. Sixty-one were treated with prone position ventilation, whereas 26 did not meet criteria. Forty-two survived (68.9%). Median (interquartile range) time from intubation to prone position ventilation was 0.28 d (0.11–0.80 d). Total prone position ventilation duration was 4.87 d (2.08–9.97 d). Prone position ventilation was applied for 30.3% (18.2–42.2%) of the first 28 days. Pao 2:Fio 2 diverged significantly by day 3 between survivors 147 (108–164) and nonsurvivors 107 (85–146), mean difference –9.632 (95% CI, –48.3 to 0.0; p = 0·05). Age, driving pressure, day 1, and day 3 Pao 2:Fio 2 were predictive of time to death. Thirty-eight (71.7%) developed ventral pressure wounds that were associated with prone position ventilation duration and day 3 Sequential Organ Failure Assessment. Limb weakness occurred in 58 (95.1%) with brachial plexus palsies in five (8.2%). Hospital-acquired infections other than central line–associated blood stream infections were infrequent. CONCLUSIONS: Prolonged prone position ventilation was feasible and relatively safe with implications for wider adoption in treating critically ill coronavirus disease 2019 patients and acute respiratory distress syndrome of other etiologies.

The master transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of antioxidant and phase II-metabolizing enzymes by activating the antioxidant response element (ARE) and thereby protects cells and tissues from oxidative stress. Pulmonary complications remain the leading cause of death in human immunodeficiency virus (HIV)-1-infected individuals, who display systemic oxidative stress and glutathione deficiency that can be modeled in transgenic rats where HIV-1-related viral proteins decrease glutathione levels and cause epithelial barrier dysfunction within the alveolar space by as yet unknown mechanisms. We hypothesized that HIV-1-related proteins inhibit Nrf2-mediated antioxidant defenses and thereby disrupt the normally tight alveolar epithelial barrier. Nrf2 RNA silencing dampened Nrf2/ARE activity, decreased the expression of the tight junction proteins zonula occludens-1, occludin, and claudin-18, increased paracellular permeability of alveolar epithelial monolayers derived from wild-type rats, and therefore reproduced the effects of HIV-1 transgene expression on the epithelial barrier that we had previously described. In contrast, upregulating Nrf2 activity, either by plasmid-mediated overexpression or treatment with the Nrf2 activator sulforaphane, increased the expression of ARE-dependent antioxidants, including NAD(P)H dehydrogenase, quinone 1 and glutathione, improved the expression of tight junction proteins, and restored the ability to form tight barriers in alveolar epithelial cells from HIV-1 transgenic rats. Taken together, these new findings argue that HIV-1-related proteins downregulate Nrf2 expression and/or activity within the alveolar epithelium, which in turn impairs antioxidant defenses and barrier function, thereby rendering the lung susceptible to oxidative stress and injury. Furthermore, this study suggests that activating the Nrf2/ARE pathway with the dietary supplement sulforaphane could augment antioxidant defenses and lung health in HIV-1-infected individuals.

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

Highlights d Efferocytosis triggers macrophages to accumulate polyamines spermidine and spermine d Polyamine accumulation is mediated by Rac1-dependent endocytic import d Inhibition of polyamine import blunts immunomodulation in response to efferocytosis