Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Lung dendritic cells in respiratory viral infection and asthma: from protection to immunopathology.

Lung dendritic cells (DCs) bridge innate and adaptive immunity, and depending on context, they also induce a Th1, Th2, or Th17 response to optimally clear infectious threats. Conversely, lung DCs can also mount maladaptive Th2 immune responses to harmless allergens and, in this way, contribute to immunopathology. It is now clear that the various aspects of DC biology can be understood only if we take into account the functional specializations of different DC subsets that are present in the lung in homeostasis or are attracted to the lung as part of the inflammatory response to inhaled noxious stimuli. Lung DCs are heavily influenced by the nearby epithelial cells, and a model is emerging whereby direct communication between DCs and epithelial cells determines the outcome of the pulmonary immune response. Here, we have approached DC biology from the perspective of viral infection and allergy to illustrate these emerging concepts.

Alveolar macrophages are epigenetically altered after inflammation, leading to long-term lung immunoparalysis.

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.

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Here, we evaluate transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurs 4 days after peak viral titer and correlates with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures.

IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease.

Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis.

Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFß negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

The cGAS-STING pathway drives type I IFN immunopathology in COVID-19.

COVID-19, which is caused by infection with SARS-CoV-2, is characterized by lung pathology and extrapulmonary complications1,2. Type I interferons (IFNs) have an essential role in the pathogenesis of COVID-19 (refs 3-5). Although rapid induction of type I IFNs limits virus propagation, a sustained increase in the levels of type I IFNs in the late phase of the infection is associated with aberrant inflammation and poor clinical outcome5-17. Here we show that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which controls immunity to cytosolic DNA, is a critical driver of aberrant type I IFN responses in COVID-19 (ref. 18). Profiling COVID-19 skin manifestations, we uncover a STING-dependent type I IFN signature that is primarily mediated by macrophages adjacent to areas of endothelial cell damage. Moreover, cGAS-STING activity was detected in lung samples from patients with COVID-19 with prominent tissue destruction, and was associated with type I IFN responses. A lung-on-chip model revealed that, in addition to macrophages, infection with SARS-CoV-2 activates cGAS-STING signalling in endothelial cells through mitochondrial DNA release, which leads to cell death and type I IFN production. In mice, pharmacological inhibition of STING reduces severe lung inflammation induced by SARS-CoV-2 and improves disease outcome. Collectively, our study establishes a mechanistic basis of pathological type I IFN responses in COVID-19 and reveals a principle for the development of host-directed therapeutics.

SARS-CoV-2 ORF8 modulates lung inflammation and clinical disease progression.

The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis.

Pulmonary inflammation and viral replication define distinct clinical outcomes in fatal cases of COVID-19.

COVID-19 has affected more than half a billion people worldwide, with more than 6.3 million deaths, but the pathophysiological mechanisms involved in lethal cases and the host determinants that determine the different clinical outcomes are still unclear. In this study, we assessed lung autopsies of 47 COVID-19 patients and examined the inflammatory profiles, viral loads, and inflammasome activation. Additionally, we correlated these factors with the patient's clinical and histopathological conditions. Robust inflammasome activation was detected in the lungs of lethal cases of SARS-CoV-2. Experiments conducted on transgenic mice expressing hACE2 and infected with SARS-CoV-2 showed that Nlrp3-/- mice were protected from disease development and lethality compared to Nlrp3+/+ littermate mice, supporting the involvement of this inflammasome in disease exacerbation. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2. Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, viral loads, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation led to different clinical outcomes. We provide important information to understand clinical variations in severe COVID-19, a process that is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

Blocking IL-17a Signaling Decreases Lung Inflammation and Improves Alveolarization in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants that is associated with life-long morbidities. Inflammatory insults contribute to BPD pathogenesis. Although the proinflammatory cytokine, IL-17a, plays a role in various neonatal inflammatory disorders, its role in BPD pathogenesis is unclear. To test the hypothesis that blocking IL-17a signaling decreases lipopolysaccharide (LPS)-mediated experimental BPD in neonatal mice, wild-type mice were injected intraperitoneally with phosphate-buffered saline or LPS during the saccular lung developmental phase. Pulmonary IL-17a expression was determined by enzyme-linked immunosorbent assay and by flow cytometry. LPS-injected mice had higher pulmonary IL-17a protein levels and IL-17a+ and IL-22+ cells. γδ T cells, followed by non-T lymphoid cells, were the primary producers of IL-17a. Wild-type mice were then injected intraperitoneally with isotype antibody (Ab) or IL-17a Ab, while they were treated with phosphate-buffered saline or LPS, followed by quantification of lung inflammatory markers, alveolarization, vascularization, cell proliferation, and apoptosis. LPS-mediated alveolar simplification, apoptosis, and cell proliferation inhibition were significantly greater in mice treated with isotype Ab than in those treated with IL-17a Ab. Furthermore, STAT1 activation and IL-6 levels were significantly greater in LPS-exposed mice treated with isotype Ab than in those treated with IL-17a Ab. The study results indicate that blocking IL-17a signaling decreases LPS-mediated experimental BPD.

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif-coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI-treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet-neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI-treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Cooperation Between Platelet ß1 and ß3 Integrins in the Arrest of Bleeding Under Inflammatory Conditions in Mice-Brief Report.

BACKGROUND: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how ß1 and ß3 integrins are involved. METHODS: The impact of isolated or combined platelet deficiency in ß1 and ß3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery. RESULTS: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-ß1-/-) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-ß3-/-) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-ß3-/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of ß1 integrins, PF4Cre-ß1-/-/ß3-/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-ß1-/-/ß3-/- platelets to inflamed microvessels. Unlike PF4Cre-ß1-/- and PF4Cre-ß3-/- mice, PF4Cre-ß1-/-/ß3-/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-ß1-/-/ß3-/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-ß3-/- animals. CONCLUSIONS: Altogether, these results show that the requirement for and degree of functional redundancy between platelet ß1 and ß3 integrins in inflammation-associated hemostasis vary with the inflammatory situation.

Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability.

BACKGROUND: Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 (calcium release-activated calcium modulator 1) is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 (Ras-proximate-1) isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation. METHODS: The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches. RESULTS: We explored Ca2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 µmol/L BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), 10 µmol/L GSK-7975A, and 5 µmol/L Gd3+. Whole-cell patch clamp measurements revealed enhanced Ca2+ release-activated Ca2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1 [C-X-C motif chemokine ligand 1], CXCL11 [C-X-C motif chemokine 11], CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1AiΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1AiΔEC mice. CONCLUSIONS: Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

Endogenous LXR signaling controls pulmonary surfactant homeostasis and prevents lung inflammation.

Lung type 2 pneumocytes (T2Ps) and alveolar macrophages (AMs) play crucial roles in the synthesis, recycling and catabolism of surfactant material, a lipid/protein fluid essential for respiratory function. The liver X receptors (LXR), LXRα and LXRß, are transcription factors important for lipid metabolism and inflammation. While LXR activation exerts anti-inflammatory actions in lung injury caused by lipopolysaccharide (LPS) and other inflammatory stimuli, the full extent of the endogenous LXR transcriptional activity in pulmonary homeostasis is incompletely understood. Here, using mice lacking LXRα and LXRß as experimental models, we describe how the loss of LXRs causes pulmonary lipidosis, pulmonary congestion, fibrosis and chronic inflammation due to defective de novo synthesis and recycling of surfactant material by T2Ps and defective phagocytosis and degradation of excess surfactant by AMs. LXR-deficient T2Ps display aberrant lamellar bodies and decreased expression of genes encoding for surfactant proteins and enzymes involved in cholesterol, fatty acids, and phospholipid metabolism. Moreover, LXR-deficient lungs accumulate foamy AMs with aberrant expression of cholesterol and phospholipid metabolism genes. Using a house dust mite aeroallergen-induced mouse model of asthma, we show that LXR-deficient mice exhibit a more pronounced airway reactivity to a methacholine challenge and greater pulmonary infiltration, indicating an altered physiology of LXR-deficient lungs. Moreover, pretreatment with LXR agonists ameliorated the airway reactivity in WT mice sensitized to house dust mite extracts, confirming that LXR plays an important role in lung physiology and suggesting that agonist pharmacology could be used to treat inflammatory lung diseases.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

In utero ventilation induces lung parenchymal and vascular alterations in extremely preterm fetal sheep.

Extremely preterm infants are often exposed to long durations of mechanical ventilation to facilitate gas exchange, resulting in ventilation-induced lung injury (VILI). New lung protective strategies utilizing noninvasive ventilation or low tidal volumes are now common but have not reduced rates of bronchopulmonary dysplasia. We aimed to determine the effect of 24 h of low tidal volume ventilation on the immature lung by ventilating preterm fetal sheep in utero. Preterm fetal sheep at 110 ± 1(SD) days' gestation underwent sterile surgery for instrumentation with a tracheal loop to enable in utero mechanical ventilation (IUV). At 112 ± 1 days' gestation, fetuses received either in utero mechanical ventilation (IUV, n = 10) targeting 3-5 mL/kg for 24 h, or no ventilation (CONT, n = 9). At necropsy, fetal lungs were collected to assess molecular and histological markers of lung inflammation and injury. IUV significantly increased lung mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) compared with CONT, and increased surfactant protein (SP)-A1, SP-B, and SP-C mRNA expression compared with CONT. IUV produced modest structural changes to the airways, including reduced parenchymal collagen and myofibroblast density. IUV increased pulmonary arteriole thickness compared with CONT but did not alter overall elastin or collagen content within the vasculature. In utero ventilation of an extremely preterm lung, even at low tidal volumes, induces lung inflammation and injury to the airways and vasculature. In utero ventilation may be an important model to isolate the confounding mechanisms of VILI to develop effective therapies for preterm infants requiring prolonged respiratory support.NEW & NOTEWORTHY Preterm infants often require prolonged respiratory support, but the relative contribution of ventilation to the development of lung injury is difficult to isolate. In utero mechanical ventilation allows for mechanistic investigations into ventilation-induced lung injury without confounding factors associated with sustaining extremely preterm lambs ex utero. Twenty-four hours of in utero ventilation, even at low tidal volumes, increased lung inflammation and surfactant protein expression and produced structural changes to the lung parenchyma and vasculature.