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Background Cysteinyl leukotrienes (CysLTs) are potent prophlogistic mediators in asthmatic patients; however, inhibition of CysLT receptor 1 is not a consistently effective treatment, suggesting additional regulatory mechanisms. Other cysteinyl-containing lipid mediators (LMs) derived from docosahexaenoic acid, namely maresin conjugates in tissue regeneration (MCTRs), were recently discovered. Therefore their production and actions in the lung are of considerable interest. Objective We sought to determine MCTR production, bioactions, and mechanisms in the human lung and in patients with experimental allergic airway inflammation. Methods LM metabololipidomic profiling of the lung was performed by using liquid chromatography with tandem mass spectrometry. Donor-derived human precision-cut lung slices were exposed to leukotriene (LT) D4, MCTRs, or both before determination of airway contraction. The actions of exogenous MCTRs on murine allergic host responses were determined in the setting of ovalbumin- and house dust mite–induced lung inflammation. Results Lipidomic profiling showed that the most abundant cysteinyl LMs in healthy human lungs were MCTRs, whereas CysLTs were most prevalent in patients with disease. MCTRs blocked LTD4-initiated airway contraction in human precision-cut lung slices. In mouse allergic lung inflammation MCTRs were present with temporally regulated production. With ovalbumin-induced inflammation, MCTR1 was most potent for promoting resolution of eosinophils, and MCTR3 potently decreased airway hyperreactivity to methacholine, bronchoalveolar lavage fluid albumin, and serum IgE levels. MCTR1 and MCTR3 inhibited lung eosinophilia after house dust mite–induced inflammation. Conclusion These results identified lung MCTRs that blocked human LTD4-induced airway contraction and promoted resolution of murine allergic airway responses when added exogenously. Together, these findings uncover proresolving mechanisms for lung responses that can be disrupted in patients with disease.

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.

Supplemental Digital Content is available in the text., Objectives: Acute respiratory distress syndrome is characterized by an overly exuberant inflammatory state in the lung. Specialized proresolving mediators are endogenous agonists for the resolution of lung inflammation and injury in health, yet their association with acute respiratory distress syndrome severity and outcomes remains to be defined. In the current study, we investigate associations between plasma levels of specialized proresolving mediators and acute respiratory distress syndrome severity and mortality. Design: Translational pilot study nested within a large prospective cohort of patients with risk factors for acute respiratory distress syndrome. Setting: ICU from a large medical center. Patients: Twenty-six Caucasian patients with acute respiratory distress syndrome and available plasma from early in critical illness. Interventions: None. Measurements and Main Results: Here, in samples from 26 acute respiratory distress syndrome patients, we examined plasma levels of select specialized proresolving mediators that promote lung injury resolution in preclinical systems, namely lipoxin A4 and maresin 1, and select prophlogistic lipid mediators linked to acute respiratory distress syndrome pathogenesis, namely cysteinyl leukotrienes and thromboxane B2. These mediators were detected by sensitive enzyme-linked immunosorbent assay: lipoxin A4 (assay range) (8.2–5,000 pg/mL), maresin 1 (7.8–1,000 pg/mL), cysteinyl leukotrienes (7.8–1,000 pg/mL), and thromboxane B2 (15.6–2,000 pg/mL). Lower plasma levels of specialized proresolving mediators were associated with increased duration of ventilatory support and ICU length of stay. Even in this small sample size, trends were evident for increased cysteinyl leukotrienes to specialized proresolving mediator ratios (cysteinyl leukotrienes/maresin 1 and cysteinyl leukotrienes/lipoxin A4) in acute respiratory distress syndrome nonsurvivors. Conclusions: Lower specialized proresolving mediator levels in acute respiratory distress syndrome patients may disrupt timely resolution of lung inflammation and/or injury and contribute to clinical severity and mortality.

Background Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. Objective We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. Methods We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FceR1γ knockdown (TRPV1Cre::FceR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. Results Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FceR1, the levels of which increase in OVA-sensitized mice. FceR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FceR1γfl/fl mice and in FceR1α–/– mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FceR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell–derived nociceptors. Conclusions Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FceR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FceR1γ expressed by nociceptors.

Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A4 (15-epi-LXA4), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R–related molecule (SIGIRR). Of interest, 15-epi-LXA4 induced A20 and SIGIRR in an lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) receptor–dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB–induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA4 also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

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., The serial assessment of the functional parameters of leukocytes isolated via an inertial microfluidic system from 50 μl of peripheral blood from sepsis patients provides significantly more prognostic information than leukocyte counting.

Summary Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.

Acute lung injury is a life-threatening condition caused by disruption of the alveolar-capillary barrier leading to edema, influx of inflammatory leukocytes, and impaired gas exchange. Specialized proresolving mediators biosynthesized from essential fatty acids, such as docosahexaenoic acid, have tissue protective effects in acute inflammation. Herein, we found that the docosahexaenoic acid–derived mediator resolvin D3 (RvD3): 4 S ,11 R ,17 S -trihydroxydocosa-5 Z ,7 E ,9 E ,13 Z ,15 E ,19 Z -hexaenoic acid was present in uninjured lungs, and increased significantly 24 to 72 hours after hydrochloric acid–initiated injury. Because of its delayed enzymatic degradation, we used aspirin-triggered (AT)-RvD3: 4 S ,11 R ,17 R -trihydroxydocosa-5 Z ,7 E ,9 E ,13 Z ,15 E ,19 Z -hexaenoic acid, a 17 R -epimer of RvD3, for in vivo experiments. Histopathological correlates of acid injury (alveolar wall thickening, edema, and leukocyte infiltration) were reduced in mice receiving AT-RvD3 1 hour after injury. AT-RvD3–treated mice had significantly reduced edema, as demonstrated by lower wet/dry weight ratios, increased epithelial sodium channel γ expression, and more lymphatic vessel endothelial hyaluronan receptor 1-positive vascular endothelial growth factor receptor 3-positive lymphatic vessels. Evidence for counterregulation of NF-κB by RvD3 and AT-RvD3 was seen in vitro and by AT-RvD3 in vivo . Increases in lung epithelial cell proliferation and bronchoalveolar lavage fluid levels of keratinocyte growth factor were observed with AT-RvD3, which also promoted cutaneous re-epithelialization. Together, these data demonstrate protective actions of RvD3 and AT-RvD3 for injured mucosa that accelerated restoration of epithelial barrier and function.

BackgroundThe incidence of high-grade immune-related adverse events (irAEs) due to immune checkpoint inhibitors (ICIs) is increasing due to the rapid expansion of indications for their use. There is an urgent need for a feasible approach of identifying patients with high-grade irAEs to ensure early detection and proper management of this unique set of toxicities.MethodsWe established one of the first inpatient services that are specifically devoted to mitigating irAEs. The service uses a multidisciplinary approach with consulting service from experts in managing irAEs. We are leveraging the electronic medical record (EMR) to triage patients who are admitted to the hospital and have received or are currently receiving ICIs. A list of patients with ICI exposure is generated daily by EMR and then curated manually to identify patients with potential irAEs.ResultsA total of 129 patients with high-grade irAEs were admitted between June 2018 and June 2019. The most common irAEs were colitis (32%), pneumonitis (30%), and hepatitis (14%). Eighty five per cent of the patients had grade 3 irAEs and 15% had grade 4–5. About half of the patients had received ICI monotherapy; 30% had received combination of ICIs and non-ICIs; and 19% had received a combination of ICIs. Only 9% of patients had steroid-refractory irAEs requiring other immunosuppressive agents. The average length of stay for irAE-related admission was 11 days with a readmission rate due to recurrent irAEs of 26% within a year.ConclusionWe demonstrated the feasibility of using the EMR to accurately triage patients with suspected irAEs to a dedicated immune-toxicity service. Our model is adaptable in major academic centers and could have a major impact on quality of care and future clinical research addressing irAEs.

e15076 Background: Immune checkpoint inhibitors (ICIs) are increasingly used in the management of cancer. High grade irAEs are uncommon but can be severe and require hospital admission. There is an urgent need for early identification and triage of patients with irAEs in order to improve their management and outcomes. Methods: We established Immunotherapy toxicity (ITOX) team as the first in nation inpatient service at DFCI and Brigham and Women's Hospital (BWH) along with our partners at Massachusetts General Hospital (MGH) that is specifically devoted to mitigating irAEs. The ITOX service is consistent of 2 PAs and a medical oncology attending with an expertise in immunotherapy. The service utilizes algorithms that are modified from the ASCO and NCCN guidelines by our medical subspecialty experts at BWH. The service uses a multi-disciplinary approach with around the hour consulting service from experts in the field including GI, pulmonary, endocrinology and others. We leveraged EPIC to triage patients who are admitted to BWH and have ever received or currently on immune checkpoint inhibitors (ICIs). The daily list generated by EPIC is then curated manually by a PA to identify patients with potential irAEs. Results: A total of 138 patients with high grade irAEs were admitted to BWH between June 2018 and June 2019. Seventy percent of the 201 irAEs- related admissions were to ITOX service (70% accuracy in triaging). Most common irAEs was colitis (31%), pneumonitis (28%) and hepatitis (13%) which is consistent with the most common reported irAEs due to ICIs. Eighty five percent of the patients had grade 3 irAEs and 15% were admitted with life threatening grade 4 adverse events. About half of the patient had received ICI monotherapy; 33% received combination of ICI and non-ICI (chemotherapy or targeted therapy) and 17% received combination of ICIs. Most patients responded to steroids and only 9% had steroid-refractory irAEs requiring other immunosuppressive agents. The average length of stay for irAEs-related admission was 11 days with readmission rate of 26% within a year. Over 50 patients consented for tissue and blood biospecimen collection at the time of toxicity. Conclusions: We demonstrated the feasibility of empowering EMR to accurately triage patients with suspected irAEs to the ITOX service that is supported by institution developed guidelines and specialists. Our model is adaptable in major academic centers and can have major impact on quality improvement and future research studies that can be conducted in this unique setting.

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. ispartof: SCIENCE IMMUNOLOGY vol:3 issue:26 ispartof: location:United States status: published

Airborne pathogens and environmental stimuli evoke immune responses in the lung. It is critical to health that these responses be controlled to prevent tissue damage and the compromise of organ function. Resolution of inflammation is a dynamic process that is coordinated by biochemical and cellular mechanisms. Recently, specialized proresolving mediators (SPMs) have been identified in resolution exudates. These molecules orchestrate anti-inflammatory and proresolving actions that are cell type specific. In this review, we highlight SPM biosynthesis, the influence of SPMs on the innate and adaptive immune responses in the lung, as well as recent insights from SPMs on inflammatory disease pathophysiology. Uncovering these mediators and cellular mechanisms for resolution is providing new windows into physiology and disease pathogenesis.

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU–NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces.

SummaryLung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8+ sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4+ and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for 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

Phospholipase D (PLD) plays important roles in cellular responses to tissue injury that are critical to acute inflammatory diseases, such as the acute respiratory distress syndrome (ARDS). We investigated the expression of PLD isoforms and related phospholipid phosphatases in patients with ARDS, and their roles in a murine model of self-limited acute lung injury (ALI). Gene expression microarray analysis on whole blood obtained from patients that met clinical criteria for ARDS and clinically matched controls (non-ARDS) demonstrated that PLD1 gene expression was increased in patients with ARDS relative to non-ARDS and correlated with survival. In contrast, PLD2 expression was associated with mortality. In a murine model of self-resolving ALI, lung Pld1 expression increased and Pld2 expression decreased 24 h after intrabronchial acid. Total lung PLD activity was increased 24 h after injury. Pld1−/− mice demonstrated impaired alveolar barrier function and increased tissue injury relative to WT and Pld2−/−, whereas Pld2−/− mice demonstrated increased recruitment of neutrophils and macrophages, and decreased tissue injury. Isoform-specific PLD inhibitors mirrored the results with isoform-specific Pld-KO mice. PLD1 gene expression knockdown in human leukocytes was associated with decreased phagocytosis by neutrophils, whereas reactive oxygen species production and phagocytosis decreased in M2-macrophages. PLD2 gene expression knockdown increased neutrophil and M2-macrophage transmigration, and increased M2-macrophage phagocytosis. These results uncovered selective regulation of PLD isoforms after ALI, and opposing effects of selective isoform knockdown on host responses and tissue injury. These findings support therapeutic strategies targeting specific PLD isoforms for the treatment of ARDS.

Specialized Pro-resolving Mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. Here, after LPS challenge, the SPMs 15-epi-lipoxin A(4) (15-epi-LXA(4)), Resolvin D1, Resolvin D2, Resolvin D3 and 17-epi-RvD1 were produced in vivo in murine lung. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and SIGIRR. Of interest, 15-epi-LXA(4) induced A20 and SIGIRR in an ALX/FPR2 receptor dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, 15-epi-LXA(4) surprisingly also enhanced pathogen clearance with increased antimicrobial peptide expression. Together, these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

290 Background: Use of immunotherapy has increased exponentially with survival benefit in many malignancies. As a result, suspected irAEs are commonly encountered, but lack of a gold standard for diagnosis puts patients at risk. We describe preliminary results of a QI project to improve accuracy of irAE diagnosis. Methods: Specialists at our institution created algorithms to define the likelihood of immune-related hepatitis, colitis, and pneumonitis based on clinical data, diagnostic results, and treatment response. Records of patients admitted from June-November 2018 with possible irAE were retrospectively reviewed by an oncology provider and an immunotoxicity specialist. A web-based tool automated irAE likelihood from clinical data based on the aglorithms. Reviewers could agree or disagree with the result and share their clinical impression. We evaluated concordance between providers and between provider and algorithm. Results: Of 65 patients, most had non-small-cell lung cancer (29%), melanoma (15%), or breast cancer (9%) and were treated with pembrolizumab (48%), nivolumab (23%), or ipilimumab/nivolumab (22%). There were 4 irAE-related deaths and 40 patients (71%) discontinued therapy for presumed toxicity. After 14 cases were excluded due to incomplete information, algorithms were applied to the remaining 51 cases (19 colitis, 8 hepatitis, 24 pneumonitis). The algorithm generated the likelihood of an irAE in 63% of reviews. Overall agreement with the algorithm was 86% (84-100% for individual algorithms), Kappa 0.8 (0.7-1.0). Overall concordance between reviewers was 76% (75-88%), Kappa of 0.7 (0.6- 0.8). Conclusions: While algorithm definitions and provider impressions were fairly consistent, cases lacking a generated likelihood suggest that some clinical scenarios are not addressed. Different interpretation of clinical information or varying experience with irAEs may explain inter-reviewer discordance, which underscores the importance of applying a standardized approach to irAE diagnosis across settings and experience levels. In the next improvement cycle, the algorithm will be refined to broaden algorithm scope, clarify criteria, and improve accuracy.

e14172 Background: Management of Immune checkpoint inhibitors (ICIs) associated irAEs requires accurate diagnosis and severity measurement. However, outside of clinical trials, there is no clear definition of irAEs to guide treatment. Methods: We established a dedicated immune toxicity inpatient service (ITox) for patients admitted with irAEs using institution-developed guidelines adapted from NCCN and ASCO. We developed definitions for ICI associated hepatitis, colitis and pneumonitis and created algorithms for defining an irAEs as definite, likely, possible, or unlikely. Algorithm fields were completed twice, by two independent reviewers including medical oncology specialists and toxicity specialists (a pulmonologist or gastroenterologist) to evaluate the accuracy of the algorithm in defining irAEs. Results: From June to November 2018, 65 patients were admitted to iTox with suspected irAEs. Various cancers patients treated with ICI were included; with lung and head and neck cancers the most common (36.9%). Pembrolizumab was the most common ICI (47.7%). ICI in combination with non-checkpoint inhibitor treatment was used in 23 (37.7%) and concurrent radiation therapy in 5 patients (7.7%). Forty (71.4%) patients discontinued ICI due to toxicity. Fifty-one patients were evaluable with irAEs: colitis (19); pneumonitis (24) and hepatitis (8). Multiple irAEs were reported in 17 patients. When applied to clinical cases, our algorithms generated a toxicity definition in 63% of cases, with which reviewers agreed most of the time (84-100%) (Kappa 0.7-1.0). Concordance between the two reviewers was 75-87% (Kappa 0.55-0.82) (Table). Conclusions: Our algorithms generated irAEs definitions with fair inter-rater reliability. These findings have real-world implications; accurate diagnosis of irAEs is essential for proper management. Defining irAEs outside the clinical trial context remains an important challenge. [Table: see text]

Negative pressure pulmonary edema (NPPE) is an important cause of noncardiogenic pulmonary edema but is rarely reported in the setting of bronchospasm. A 43-year-old woman with severe reactive airway disease suffered an episode of severe bronchospasm after endotracheal extubation following an otherwise uneventful general anesthetic. Subsequently, she developed clinical and radiographic signs of pulmonary edema in the absence of other symptoms of acute left-sided heart failure, leading to the diagnosis of noncardiogenic pulmonary edema. She received noninvasive positive pressure ventilation for a few hours, after which her clinical and radiologic signs and symptoms of pulmonary edema were greatly improved. This clinical scenario strongly suggests NPPE. We submit that it is possible to create NPPE by generating highly negative intrathoracic pressures in the setting of severe bronchospasm.

Type 2 innate lymphoid cells (ILC2s) are important for both mucosal homeostasis as well as for initiation and progression of allergy and asthma. Alarmin cytokines produced by damaged or stressed epithelial cells activate ILC2s, however, the role of other pathways regulating the homeostatic versus inflammatory functions of ILC2s remain poorly defined. We generated transcriptional profiles of thousands of lung-resident ILCs isolated from mice challenged with either PBS or the alarmin cytokines IL-25 or IL-33 using massively parallel droplet-based single-cell RNA sequencing (scRNA-seq). We found significant transcriptional heterogeneity within activated ILCs and identified differentially expressed genes as potential novel regulators of ILC function. One of the genes, neuromedin U receptor 1 (NMUR1), is a receptor for the neuropeptide neuromedin U (NMU) and is specifically expressed by ILC2s and not by other immune cells. Nmur1 is particularly expressed by ILC2s at steady-state and after IL-25 treatment. In vivo NMU synergized with IL-25 to induce more severe airway inflammation than was seen with either IL-25 or NMU alone and this effect was dependent on ILCs. Mechanistically, IL-25 + NMU expanded highly proliferative and inflammatory ILCs with distinct transcriptional profiles and increased expression of IL-5 and IL-13. We observed reduced ILC2 frequency and function in NMU- or NMUR1-deficient mice after allergen challenge and found that ILCs from these mice had a lower score for an inflammatory ILC2 gene signature. Our data therefore establish a novel neuro-immune pathway that amplifies ILC2-driven airway inflammation.

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly-GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.

Bacterial pneumonia is a leading cause of morbidity and mortality worldwide. Host responses to contain infection and mitigate pathogen-mediated lung inflammation are critical for pneumonia resolution. Aspirin-triggered resolvin D1 (AT-RvD1; 7S,8R,17R trihydroxy-4Z,9E,11E,13Z,15E,19Z docosahexaenoic acid) is a lipid mediator that displays organ protective actions in sterile lung inflammation, and regulates pathogen-initiated cellular responses. Here, in a self-resolving murine model of Escherichia coli pneumonia, lipid mediator metabololipidomics performed on lungs obtained at baseline, 24 hours and 72 hours after infection uncovered temporal regulation of endogenous AT-RvD1 production. Early treatment with exogenous AT-RvD1 (1 hr post-infection) enhanced clearance of E.coli and Pseudomonas aeruginosa in vivo, and lung macrophage phagocytosis of fluorescent bacterial particles ex vivo. Characterization of macrophage subsets in the alveolar compartment during pneumonia identified efferocytosis by infiltrating macrophages (CD11bHi CD11cLow) and exudative macrophages (CD11bHi CD11cHi). AT-RvD1 increased efferocytosis by these cells ex vivo, and accelerated neutrophil clearance during pneumonia in vivo. These anti-bacterial and pro-resolving actions of AT-RvD1 were additive to antibiotic therapy. Taken together, these findings suggest that the pro-resolving actions of AT-RvD1 during pneumonia represent a novel host-directed therapeutic strategy to complement the current antibiotic centered approach to combatting infections.

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that LyGDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.

Asthma is a chronic inflammatory disease that fails to resolve. Recently, a key role for type 2 innate lymphoid cells (ILC2s) was linked to asthma pathogenesis; however, mechanisms for ILC2 regulation remain to be determined. In this study, metabololipidomics of murine lungs identified temporal changes in endogenous maresin 1 (MaR1) during self-limited allergic inflammation. Exogenous MaR1 reduced lung inflammation and ILC2 expression of IL-5 and IL-13 and increased amphiregulin. MaR1 augmented de novo generation of regulatory T cells (Tregs), which interacted with ILC2s to markedly suppress cytokine production in a TGF-β–dependent manner. Ab-mediated depletion of Tregs interrupted MaR1 control of ILC2 expression of IL-13 in vivo. Together, the findings uncover Tregs as potent regulators of ILC2 activation; MaR1 targets Tregs and ILC2s to restrain allergic lung inflammation, suggesting MaR1 as the basis for a new proresolving therapeutic approach to asthma and other chronic inflammatory diseases.

Bacteria induced pneumonia remains a major global threat to health. Even with effective antibiotic treatment, the host response can be exuberant, resulting in tissue damage and morbidity. Resolution of inflammation is an active process governed in part by specialized pro-resolving mediators (SPMs). We hypothesized that the SPM, aspirin triggered lipoxin A4 (ATL) engaged airway mucosal epithelial cells to enhance resolution. In a murine model of bacterial aspiration pneumonia for E. coli, ATL (100 ng, iv) decreased the lung bacterial burden in an additive manner with ciprofloxacin (0.2 mg/kg, ip). Further, ATL also significantly limited neutrophils recruitment and decreased pro-inflammatory cytokine (IL-1α) and chemokine production (KC). These ATL mediated protective responses were mediated in part via regulation of NF-kB activation. In response to LPS and E. coli in vitro, ATL selectively influenced airway epithelial cells expression of NF-kB regulators A20, SIGIRR and ST2. In an in vitro bacteria killing assay by airway epithelial cells, ATL directly limited bacterial growth by increasing select antimicrobial peptides expression (LL37 and BPI). mCRAMP deficient mice (human homolog of LL37) failed to limit lung bacterial growth in E. coli pneumonia and ATL treatment was significantly less efficient in promoting host defense in these mice. In conclusion, the SPM ATL can limit pathogen mediated mucosal inflammation and enhance host defense in pneumonia via the selective regulation of NF-κB and antimicrobial peptides in airway epithelial cells. These findings with ATL provide evidence for SPM targeted treatment of the host response to pneumonia that has the potential for therapeutic benefit in combination with antibiotics.

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.

Purpose The study aims at studying the ophthalmologic status in a group of thalassemia patients. Design A cross-sectional study. Methods Eighty-four thalassemia patients were randomly selected and underwent an ophthalmologic examination. Results Visual acuity (VA) was affected in 13 patients and retinal pigment epithelial changes were detected in 21 patients. Decrease in VA was found to be significantly associated with type of thalassemia ( P P = .05). Retinal pigment epithelium changes, conversely, were significantly associated with type of iron chelation. Conclusions The occurrence of ophthalmologic problems in Lebanese thalassemic patients is common. These patients, especially those with severe anemia, should be screened for such complications. However, the results were not conclusive on whether these complications are attributable to the anemia, the iron overload, or a side effect of the iron chelating agents.

Rationale: Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation. Objectives: This study investigated changes in lung NOS expression and activity in a mouse model of ventilator-induced lung injury. Methods: C57BL/6J (wild-type [WT]) and iNOS-deficient (iNOS−/−) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay. Results: iNOS mRNA and protein expression was absent in iNOS−/− mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS−/− mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS−/− mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS−/− mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS−/− mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine. Conclusion: Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury.