The Maresin 1-LGR6 axis decreases respiratory syncytial virus-induced lung inflammation.

The resolution of infection is an active process with specific molecular and cellular mechanisms that temper inflammation and enhance pathogen clearance. Here, the specialized pro-resolving mediator (SPM) Maresin 1 (MaR1) inhibited respiratory syncytial virus (RSV)-induced inflammation. inlerleukin-13 production from type 2 innate lymphoid cells (ILC) and CD4 T helper type 2 cells was decreased by exogenous MaR1. In addition, MaR1 increased amphiregulin production and decreased RSV viral transcripts to promote resolution. MaR1 also promoted interferon-ß production in mouse lung tissues and also in pediatric lung slices. MaR1 significantly inhibited the RSV-triggered aberrant inflammatory phenotype in FoxP3-expressing Tregs. The receptor for MaR1, leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), was constitutively expressed on Tregs. Following RSV infection, mice lacking Lgr6 had exacerbated type 2 immune responses with an increased viral burden and blunted responses to MaR1. Together, these findings have uncovered a multi-pronged protective signaling axis for MaR1-Lgr6, improving Tregs's suppressive function and upregulating host antiviral genes resulting in decreased viral burden and pathogen-mediated inflammation, ultimately promoting restoration of airway mucosal homeostasis.

15-epi-lipoxin A5 promotes neutrophil exit from exudates for clearance by splenic macrophages.

Specialized proresolving mediators (SPMs) promote local macrophage efferocytosis but excess leukocytes early in inflammation require additional leukocyte clearance mechanism for resolution. Here, neutrophil clearance mechanisms from localized acute inflammation were investigated in mouse dorsal air pouches. 15-HEPE (15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid) levels were increased in the exudates. Activated human neutrophils converted 15-HEPE to lipoxin A5 (5S,6R,15S-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), 15-epi-lipoxin A5 (5S,6R,15R-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), and resolvin E4 (RvE4; 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid). Exogenous 15-epi-lipoxin A5, 15-epi-lipoxin A4 and a structural lipoxin mimetic significantly decreased exudate neutrophils and increased local tissue macrophage efferocytosis, with comparison to naproxen. 15-epi-lipoxin A5 also cleared exudate neutrophils faster than the apparent local capacity for stimulated macrophage efferocytosis, so the fate of exudate neutrophils was tracked with CD45.1 variant neutrophils. 15-epi-lipoxin A5 augmented the exit of adoptively transferred neutrophils from the pouch exudate to the spleen, and significantly increased splenic SIRPa+ and MARCO+ macrophage efferocytosis. Together, these findings demonstrate new systemic resolution mechanisms for 15-epi-lipoxin A5 and RvE4 in localized tissue inflammation, which distally engage the spleen to activate macrophage efferocytosis for the clearance of tissue exudate neutrophils.

Specialized pro-resolving mediators in respiratory diseases.

PURPOSE OF REVIEW: Persistent unresolved inflammation results in a number of pathologic respiratory diseases including asthma, cystic fibrosis, acute respiratory distress syndrome (ARDS) and coronavirus disease 2019 (COVID-19)-associated ARDS. Inflammation resolution is an active series of biologic processes orchestrated by a family of bioactive specialized pro-resolving mediators (SPMs) derived from essential omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). In this review, we highlight recent findings on dysregulated inflammation resolution in common respiratory diseases and recent literature on SPM generation with PUFA dietary supplementation with relevance to diseases of respiratory inflammation. RECENT FINDINGS: Human studies and preclinical models of diseases of lung inflammation have revealed disequilibrium in the levels of pro-inflammatory versus pro-resolving mediators. Recent studies identified actions for SPMs on regulating prophlogistic host responses and stimulating inflammation resolution pathways in inflammatory respiratory diseases. SUMMARY: Dietary marine oils are enriched in PUFAs and contain parent omega-3 and omega-6 fatty acids and precursors for conversion to SPMs. Nutritional supplementation with fish oils can boost SPM levels and offer a therapeutic approach targeting inflammation resolution pathways for diseases of lung inflammation.

Human NK Cell Cytoskeletal Dynamics and Cytotoxicity Are Regulated by LIM Kinase.

NK cells provide immune surveillance and host protection against viruses and tumors through their cytotoxic effector function. Cytoskeletal rearrangement is necessary for NK cell lytic granule trafficking and immune synapse formation to trigger apoptosis of targeted cells. LIM kinase (LIMK) regulates F-actin remodeling by phosphorylating cofilin to inhibit actin severing and depolymerization. In this study, in human NK cells, the glucocorticoid dexamethasone downregulated LIMK expression, F-actin accumulation at the immune synapse, lytic granule trafficking, and cytotoxicity. In contrast, the specialized proresolving mediator lipoxin A4 promoted NK cell LIMK expression, lytic granule polarization to the immune synapse and cytotoxicity. Using a LIMK inhibitor, we show that LIMK activity is necessary for NK cell cytotoxicity, including lipoxin A4's proresolving actions. Together, our findings identify LIMK as an important control mechanism for NK cell cytoskeletal rearrangement that is differentially regulated by glucocorticoids and specialized proresolving mediators to influence NK cell cytotoxicity.

Invasive and noninvasive ventilation strategies for acute respiratory failure in children with coronavirus disease 2019.

PURPOSE OF REVIEW: Severe Acute Respiratory Syndrome Coronavirus 2 presents as symptomatic coronavirus disease 2019 (COVID-19) disease in susceptible patients. Severe pediatric COVID-19 disease is rare, limiting potential data accumulation on associated respiratory failure in children. Pediatric intensivists and pulmonologists managing COVID-19 patients look to adult guidelines and pediatric-specific consensus statements to guide management. The purpose of this article is to review the current literature and recommended strategies for the escalation of noninvasive and invasive respiratory support for acute respiratory failure associated with COVID-19 disease in children. RECENT FINDINGS: There are no prospective studies comparing COVID-19 treatment strategies in children. Adult and pediatric ventilation management interim guidance is based on evidence-based guidelines in non-COVID acute respiratory distress syndrome, with considerations of (1) noninvasive positive pressure ventilation versus high-flow nasal cannula and (2) high versus lower positive end expiratory pressure strategies related to lung compliance and potential lung recruitability. SUMMARY: Management of acute respiratory failure from COVID-19 requires individualized titration of noninvasive and invasive ventilation modalities with consideration of preserved or compromised pulmonary compliance. Research regarding best practices in the management of pediatric severe COVID-19 with respiratory failure is lacking and is acutely needed as the pandemic surges and vaccination of the pediatric population will be delayed compared to adults.

Non-type 2 inflammation in severe asthma is propelled by neutrophil cytoplasts and maintained by defective resolution.

Asthma is a highly prevalent heterogeneous inflammatory disorder of the airways. Not all patients respond to anti-inflammatory treatment with corticosteroids, leading to significant morbidity in severe asthma. Much attention has been paid to defining the cellular and molecular mechanisms of type 2 inflammation that are operative in asthma. Development of targeted therapies for pathologic type 2 inflammation is opening a new approach to asthma treatment; however, not all asthmatics have type 2 airway inflammation, especially those with severe corticosteroid-refractory asthma. Much less is known about non-type 2 immunological mechanisms in asthma. In health, inflammation triggers resolution mechanisms that control immune (type 1 and type 2) responses and enable the restoration of tissue homeostasis. The resolution response is comprised of cellular and molecular events, including production of specialized pro-resolving mediators (SPMs). SPMs halt leukocyte recruitment, promote macrophage efferocytosis, and restore epithelial barrier integrity, all of which are critical to resolution of inflammation in the lungs. Here, we review recent insights into the disruption of these homeostatic mechanisms and their contributions to non-type 2 inflammation in severe asthma immunopathogenesis.

Pediatric Respiratory Syncytial Virus Hospitalizations and Respiratory Support After the COVID-19 Pandemic.

Importance: After the COVID-19 pandemic, there was a surge of pediatric respiratory syncytial virus (RSV) infections, but national data on hospitalization and intensive care unit use and advanced respiratory support modalities have not been reported. Objective: To analyze demographics, respiratory support modes, and clinical outcomes of children with RSV infections at tertiary pediatric hospitals from 2017 to 2023. Design, Setting, and Participants: This cross-sectional study evaluated children from 48 freestanding US children's hospitals registered in the Pediatric Health Information System (PHIS) database. Patients 5 years or younger with RSV from July 1, 2017, to June 30, 2023, were included. Each season was defined from July 1 to June 30. Prepandemic RSV seasons included 2017 to 2018, 2018 to 2019, and 2019 to 2020. The postpandemic season was delineated as 2022 to 2023. Exposure: Hospital presentation with RSV infection. Main Outcomes and Measures: Data on emergency department presentations, hospital or intensive care unit admission and length of stay, demographics, respiratory support use, mortality, and cardiopulmonary resuscitation were analyzed. Postpandemic season data were compared with prepandemic seasonal averages. Results: A total of 288 816 children aged 5 years or younger (median [IQR] age, 8.9 [3.3-21.5] months; 159 348 [55.2%] male) presented to 48 US children's hospitals with RSV from July 1, 2017, to June 30, 2023. Respiratory syncytial virus hospital presentations increased from 39 698 before the COVID-19 pandemic to 94 347 after the pandemic (P < .001), with 86.7% more hospitalizations than before the pandemic (50 619 vs 27 114; P < .001). In 2022 to 2023, children were older (median [IQR] age, 11.3 [4.1-26.6] months vs 6.8 [2.6-16.8] months; P < .001) and had fewer comorbidities (17.6% vs 21.8% of hospitalized patients; P < .001) than during prepandemic seasons. Advanced respiratory support use increased 70.1% in 2022 to 2023 (9094 vs 5340; P < .001), and children requiring high-flow nasal cannula (HFNC) or noninvasive ventilation (NIV) were older than during prepandemic seasons (median [IQR] age for HFNC, 6.9 [2.7-16.0] months vs 4.6 [2.0-11.7] months; for NIV, 6.0 [2.1-16.5] months vs 4.3 [1.9-11.9] months). Comorbid conditions were less frequent after the pandemic across all respiratory support modalities (HFNC, 14.9% vs 19.1%, NIV, 22.0% vs 28.5%, invasive mechanical ventilation, 30.5% vs 38.0%; P < .001). Conclusions and Relevance: This cross-sectional study identified a postpandemic pediatric RSV surge that resulted in markedly increased hospital volumes and advanced respiratory support needs in older children with fewer comorbidities than prepandemic seasons. These clinical trends may inform novel vaccine allocation to reduce the overall burden during future RSV seasons.

Vitamin D constrains inflammation by modulating the expression of key genes on Chr17q12-21.1.

Vitamin D possesses immunomodulatory functions and vitamin D deficiency has been associated with the rise in chronic inflammatory diseases, including asthma (Litonjua and Weiss, 2007). Vitamin D supplementation studies do not provide insight into the molecular genetic mechanisms of vitamin D-mediated immunoregulation. Here, we provide evidence for vitamin D regulation of two human chromosomal loci, Chr17q12-21.1 and Chr17q21.2, reliably associated with autoimmune and chronic inflammatory diseases. We demonstrate increased vitamin D receptor (Vdr) expression in mouse lung CD4+ Th2 cells, differential expression of Chr17q12-21.1 and Chr17q21.2 genes in Th2 cells based on vitamin D status and identify the IL-2/Stat5 pathway as a target of vitamin D signaling. Vitamin D deficiency caused severe lung inflammation after allergen challenge in mice that was prevented by long-term prenatal vitamin D supplementation. Mechanistically, vitamin D induced the expression of the Ikzf3-encoded protein Aiolos to suppress IL-2 signaling and ameliorate cytokine production in Th2 cells. These translational findings demonstrate mechanisms for the immune protective effect of vitamin D in allergic lung inflammation with a strong molecular genetic link to the regulation of both Chr17q12-21.1 and Chr17q21.2 genes and suggest further functional studies and interventional strategies for long-term prevention of asthma and other autoimmune disorders.

Mouse phospholipid phosphatase 6 regulates dendritic cell cholesterol, macropinocytosis, and allergen sensitization.

Lipid phosphate phosphatases are a family of enzymes with diverse cellular metabolic functions. Phospholipid phosphatase 6 (PLPP6) is a regulator of cellular polyisoprenyl phosphates; however, its in vivo functions remain to be determined. Here, mouse PLPP6 was characterized to possess similar catalytic properties as the human enzyme. Plpp6 knockout mice (Plpp6 -/- ) were generated and displayed decreased airway allergen sensitization, pointing to a role for PLPP6 in the early events of lung allergic responses. Dendritic cell (DC) responses were investigated and endocytosis of allergen via macropinocytosis was decreased in Plpp6 -/- DCs that had lower cholesterol content. When reversed by cholesterol loading, the DC macropinocytosis defect is corrected. Adoptive transfer of Plpp6 -/- DCs to wild-type mice during sensitization was sufficient to decrease allergen-induced responses. Together, our findings have identified PLPP6 as a pivotal regulator of DC cholesterol content and macropinocytosis, cellular mechanisms that are important for pathologic responses in allergen-induced lung inflammation.

Inflammation resolution circuits are uncoupled in acute sepsis and correlate with clinical severity.

Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized proresolving mediators are agonists for antiinflammation and for promoting resolution, and they are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized proresolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and patients with sepsis by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte proresolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.

Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity.

Dysregulated leukocyte responses underlie the pathobiology of sepsis, which is a leading cause of death. However, measures of leukocyte function are not routinely available in clinical care. Here we report the development and testing of an inertial microfluidic system for the label-free isolation and downstream functional assessment of leukocytes from 50 µl of peripheral blood. We used the system to assess leukocyte phenotype and function in serial samples from 18 hospitalized patients with sepsis and 10 healthy subjects. The sepsis samples had significantly higher levels of CD16dim and CD16- neutrophils and CD16+ 'intermediate' monocytes, as well as significantly lower levels of neutrophil-elastase release, O2- production and phagolysosome formation. Repeated sampling of sepsis patients over 7 days showed that leukocyte activation (measured by isodielectric separation) and leukocyte phenotype and function were significantly more predictive of the clinical course than complete-blood-count parameters. We conclude that the serial assessment of leukocyte function in microlitre blood volumes is feasible and that it provides significantly more prognostic information than leukocyte counting.

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4(+) T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4(+) T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4(+) T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4(+) T cells. Despite this, we found that HIV-2-specific CD4(+) T cells contained more viral DNA than memory CD4(+) T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4(+) T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4(+) T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4(+) T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4(+) T-cell help in vivo.

Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma.

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4+ T cells. Bronchoalveolar lavage fluid from patients with severe asthma and high neutrophil counts had detectable NETs and cytoplasts that were positively correlated with IL-17 levels. Together, these translational findings have identified neutrophil cytoplast formation in asthmatic lung inflammation and linked the cytoplasts to T helper 17-mediated neutrophilic inflammation in severe asthma.

Bronchoprotective mechanisms for specialized pro-resolving mediators in the resolution of lung inflammation.

Bronchi are exposed daily to irritants, microbes and allergens as well as extremes of temperature and acid. The airway mucosal epithelium plays a pivotal role as a sentinel, releasing alarmins when danger is encountered. To maintain homeostasis, an elaborate counter-regulatory network of signals and cellular effector mechanisms are needed. Specialized pro-resolving mediators (SPMs) are chemical mediators that enact resolution programs in response to injury, infection or allergy. SPMs are enzymatically derived from essential polyunsaturated fatty acids with potent cell-type specific immunoresolvent properties. SPMs signal by engaging cell-based receptors to turn off acute inflammatory responses and restore tissue homeostasis. Several common lung diseases involving the airways, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF), are characterized by unresolved bronchial inflammation. In preclinical murine models of lung disease, SPMs carry potent bronchoprotective actions. Here, we review cellular and molecular effects for SPM-initiated catabasis in the lung and their human translation.

Natural killer cell-mediated inflammation resolution is disabled in severe asthma.

Severe asthma is typically characterized by chronic airway inflammation that is refractory to corticosteroids and associated with excess morbidity. Patients were recruited into the National Heart, Lung, and Blood Institute-sponsored Severe Asthma Research Program and comprehensively phenotyped by bronchoscopy. Bronchoalveolar lavage (BAL) cells were analyzed by flow cytometry. Compared with healthy individuals (n = 21), patients with asthma (n = 53) had fewer BAL natural killer (NK) cells. Patients with severe asthma (n = 29) had a marked increase in the ratios of CD4+ T cells to NK cells and neutrophils to NK cells. BAL NK cells in severe asthma were skewed toward the cytotoxic CD56dim subset, with significantly increased BAL fluid levels of the cytotoxic mediator granzyme A. The numbers of BAL CD56dim NK cells and CCR6-CCR4- T helper 1-enriched CD4+ T cells correlated inversely with lung function [forced expiratory volume in 1 s (FEV1) % predicted] in asthma. Relative to cells from healthy controls, peripheral blood NK cells from asthmatic patients had impaired killing of K562 myeloid target cells despite releasing more cytotoxic mediators. Ex vivo exposure to dexamethasone markedly decreased blood NK cell lysis of target cells and cytotoxic mediator release. NK cells expressed airway lipoxin A4/formyl peptide receptor 2 receptors, and in contrast to dexamethasone, lipoxin A4-exposed NK cells had preserved functional responses. Together, our findings indicate that the immunology of the severe asthma airway is characterized by decreased NK cell cytotoxicity with increased numbers of target leukocytes, which is exacerbated by corticosteroids that further disable NK cell function. These failed resolution mechanisms likely contribute to persistent airway inflammation in severe asthma.

ALX receptor ligands define a biochemical endotype for severe asthma.

BACKGROUND: In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). METHODS: ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). RESULTS: Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. CONCLUSIONS: Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. TRIAL REGISTRATION: Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826)FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

DHA- and EPA-derived resolvins, protectins, and maresins in airway inflammation.

Essential fatty acids can serve as important regulators of inflammation. A new window into mechanisms for the resolution of inflammation was opened with the identification and structural elucidation of mediators derived from these fatty acids with pro-resolving capacity. Inflammation is necessary to ensure the continued health of the organism after an insult or injury; however, unrestrained inflammation can lead to injury "from within" and chronic changes that may prove both morbid and fatal. The resolution phase of inflammation, once thought to be a passive event, is now known to be a highly regulated, active, and complex program that terminates the inflammatory response once the threat has been contained. Specialized pro-resolving mediators (SPMs) are biosynthesized from omega-3 essential fatty acids to resolvins, protectins, and maresins and from omega-6 fatty acids to lipoxins. Through cell-specific actions mediated through select receptors, these SPMs are potent regulators of neutrophil infiltration, cytokine and chemokine production, and clearance of apoptotic neutrophils by macrophages, promoting a return to tissue homeostasis. This process appears to be defective in several common human lung diseases, such as asthma and COPD, which are characterized by chronic unrestrained inflammation and significant associated morbidity. Here, we highlight translational research in animal models of disease and with human subjects that sheds light on this rapidly evolving area of science and review the molecular and cellular components of the resolution of lung inflammation.