Type 2 (T2) immune cells dominate the airways of patients with mild-moderate asthma (MMA) with a more complex type 1 (T1)-T2 mixed immune response evident in treatment-refractory severe asthma (SA). We hypothesized that comparing the transcriptomes of the airway epithelium of patients with SA and MMA would reveal molecular signatures associated with more severe disease in the context of a complex immune response. Using our interpretable machine learning tool, SLIDE, meaningful latent factors (context-specific gene co-expression networks) were revealed that distinguished SA from MMA. Unexpectedly, an aberrant high expression of normally host-protective, membrane-tethered, and IFN-inducible mucins, MUC1 and MUC4, was identified in SA. Gene networks in the significant latent factors discriminating SA from MMA corresponded to enrichment of a keratinization program in SA airways. Keratinization was marked by increased expression of the stress keratin KRT16, signifying squamous metaplasia suggesting adaptive reprogramming of the airway epithelium in response to chronic stress. These mucins and KRT16 were inversely associated with lung function in 2 separate asthma cohorts. Imaging of endobronchial biopsies revealed significantly higher KRT16 protein expression in SA compared with MMA that strongly correlated with MUC1 protein expression. Our study identifies dysregulated host-protective and maladaptive repair responses in SA distinguishing from MMA.

An early event after lung injury is extracellular matrix (ECM) remodeling and the formation of a provisional matrix. While megakaryocytes and platelets (Mgk/plt) play important roles in hemostasis, their impact on the matrix in lung injury is not fully understood. Using lung intravital microscopy, 3D large area scanning with multiphoton confocal hybrid imaging, and label-free quantitative proteomics, the matricellular protein thrombospondin-1 (TSP1) arising from Mgk/plt protects the lung from alveolar injury. Mgk/plt cell-specific Thbs1 knockout (cKO) mice show increased alveolar barrier disruption with exaggerated neutrophil-mediated injury. The cKO mice exhibit striking extracellular matrix re-organization with reduction in basement membrane matrix protein COL6A1 after injury. Moreover, cKO mice increase Mgk numbers to regions of fibrillar collagen deposition and alveolar leak. Our findings indicate Mgk/plt-derived TSP1 represents a key mechanism of matrix stabilization by protecting the basement membrane from neutrophil-mediated proteolytic damage, regulating injury severity and Mgk numbers at the alveolar interface.

Allergic asthma is driven by type 2 immune responses, including type 2 innate lymphoid cells (ILC2s). Although ILC2s are activated by the tissue alarmins interleukin (IL)-33 and IL-25, these signals do not intrinsically enforce type 2 identity and the mechanisms that maintain type 2 cytokine expression remain unclear. Here we show that allergen-induced IL-33 and IL-25 rapidly induce IL-9, which in turn upregulates the transcriptional repressor Blimp-1 in ILC2s. Blimp-1 sustains type 2 immunity by directly repressing type 1 inflammatory programs, including expression of interferon-γ and tumor necrosis factor. Deletion of Blimp-1 in ILC2s increased type 1 cytokine production and reduced IL-5 and IL-13 expression, eosinophil recruitment and mucus production in the lung. In contrast, IL-9 expression was enhanced in the absence of Blimp-1, leading to increased mast cell recruitment. Together, these findings identify Blimp-1 as a key regulator of ILC2 transcriptional fidelity that stabilizes type 2 inflammation while constraining divergent inflammatory programs during allergic responses.

Aberrant immune response is a hallmark of asthma, with 5%-10% of patients suffering from severe disease exhibiting poor response to standard treatment. A better understanding of the immune responses contributing to disease heterogeneity is critical for improving asthma management. T cells are major players in the orchestration of asthma, in both mild and severe disease, but it is unclear whether specific T cell subsets influence asthma symptom duration. Here we show a significant association of airway CD8+ effector memory T cells re-expressing CD45RA (TEMRAs), but not CD8+CD45RO+ or tissue-resident memory T cells, with asthma duration in patients with severe asthma (SA) but not mild to moderate asthma (MMA). Higher frequencies of IFN-γ+CD8+ TEMRAs compared with IFN-γ+CD45RO+ T cells were detected in SA airways, and the TEMRAs from patients with SA but not MMA proliferated ex vivo, although both expressed cellular senescence-associated biomarkers. Prompted by the transcriptomic profile of SA CD8+ TEMRAs and proliferative response to IL-15, airway IL15 expression was higher in patients with SA compared with MMA. Additionally, IL15 expression in asthmatic airways negatively correlated with lung function. Our findings add what we believe is a new dimension to understanding asthma heterogeneity, identifying IL-15 as a potential target for treatment.

OBJECTIVES:Soluble ST2 (sST2), a decoy receptor for the alarmin interleukin-33 (IL-33), has been implicated in adverse clinical outcomes in acute respiratory failure (ARF). We evaluated sST2 distribution across diverse cohorts of patients with different etiologies of ARF, compared plasma and lower respiratory tract (LRT) concentrations, and examined associations with individual organ dysfunction, biological subphenotypes, and outcomes. DESIGN:Observational study. SETTING:Multicenter cohorts of ARF patients. PATIENTS:A total of 1432 ARF patients, including 863 non-COVID and 569 COVID-19 cases, from five cohorts. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:sST2 levels were measured in plasma and LRT specimens (when available) and analyzed for associations with ARF etiology, severity, organ dysfunction, systemic host response, subphenotypes, and 30-day mortality. Plasma sST2 levels were higher in non-COVID ARF patients compared with COVID-19 patients ( p < 0.05) and were markedly elevated compared with LRT levels (> 19-fold), with weak intercompartmental correlation. Elevated plasma sST2 levels were associated with extrapulmonary organ dysfunction and a hyperinflammatory ARF subphenotype but not with respiratory indices, including hypoxemia. Plasma sST2 independently predicted 30-day mortality in pooled cohort data, adjusted for age, sex, and illness severity. In longitudinal measurements, nonsurvivors had persistently elevated plasma sST2 levels in the first 2 weeks of critical illness compared with survivors. CONCLUSIONS:Plasma sST2 levels independently predict outcomes in ARF and are strongly associated with extrapulmonary organ dysfunction. The weak correlation between plasma and LRT sST2 levels suggests a predominantly systemic source. These findings highlight the potential of the IL-33/ST2 axis as a therapeutic target and warrant further investigation into its role in multiple organ dysfunction in ARF.

Asthma is orchestrated by an aberrant immune response involving a complex interplay between multiple inflammatory cell types. An increase in Th2 cells in the asthmatic airway is a hallmark of asthma, and biologics blocking their effector functions have been life-changing for many severe asthma patients who poorly respond to immunosuppression by corticosteroids. However, studies in the past decade have highlighted not only other cell types that also produce Th2 cytokines boosting the Type 2/T2 phenotype but also a heightened IFN-γ response, primarily from T cells, referred to as a Type 1/T1 immune response. Data derived from studies of immune cells in the airways and mouse models of severe asthma suggest a role of IFN-γ in corticosteroid resistance, airway hyperreactivity, and also airway remodeling via effects on other cell types including mast cells, eosinophils, airway epithelial cells, and airway smooth muscle cells. The simultaneous presence of T1 and T2 immune responses is detectable in the sickest of asthma patients in whom corticosteroids suppress the T2 but not the T1 response. This article has reviewed our current understanding of the complex T1-T2 interplay in severe asthma highlighting mediators that impact both arms which may be targeted alone or in combination for disease alleviation.

Asthma is a chronic inflammatory disease of the airways long known for phenotypic heterogeneity. Phenotyping studies in asthma have led to a better characterization of disease pathogenesis, yet further work is needed to pair available treatments with disease endotypes. In this review, the biology of targeted pathways is discussed along with the efficacy of biologic therapies targeting those pathways. Results of asthma clinical trials are included, as well as results of trials in related diseases. This review then analyzes how biologics help to inform the complex immunobiology of asthma and further guide their use while identifying areas for future research.

Klebsiella pneumoniae (KP) is an extracellular Gram-negative bacterium that causes infections in the lower respiratory and urinary tracts and the bloodstream. STAT1 is a master transcription factor that acts to maintain T cell quiescence under homeostatic conditions. Although STAT1 helps defend against systemic spread of acute KP intrapulmonary infection, whether STAT1 regulation of T cell homeostasis impacts pulmonary host defense during acute bacterial infection and injury is less clear. Using a clinical KP respiratory isolate and a pneumonia mouse model, we found that STAT1 deficiency led to an early neutrophil-dominant transcriptional profile and neutrophil recruitment in the lung preceding widespread bacterial dissemination and lung injury development. Yet, myeloid cell STAT1 was dispensable for control of KP proliferation and dissemination, because myeloid cell-specific STAT1-deficient (LysMCre/WT;Stat1fl/fl) mice showed bacterial burden in the lung, liver, and kidney similar to that of their wild-type littermates. Surprisingly, IL-17-producing CD4+ T cells infiltrated Stat1-/- murine lungs early during KP infection. The increase in Th17 cells in the lung was not due to preexisting immunity against KP and was consistent with circulating rather than tissue-resident CD4+ T cells. However, blocking global IL-17 signaling with anti-IL-17RC administration led to increased proliferation and dissemination of KP, suggesting that IL-17 provided by other innate immune cells is essential in defense against KP. Contrastingly, depletion of CD4+ T cells reduced Stat1-/- murine lung bacterial burden, indicating that early CD4+ T cell activation in the setting of global STAT1 deficiency is pathogenic. Altogether, our findings suggest that STAT1 employs myeloid cell-extrinsic mechanisms to regulate neutrophil responses and provides protection against invasive KP by restricting nonspecific CD4+ T cell activation and immunopathology in the lung.

A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)−1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes.

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure. This study reports training by lipopolysaccharide to expand neutrophils expressing the anti-bacterial galectin-3 protein defending mice from a lethal bacterial infection, a similar signature associated with survivors of respiratory failure in humans.

The severity of bacterial pneumonia can be worsened by impaired innate immunity resulting in ineffective pathogen clearance. We describe a mitochondrial protein, aspartyl-tRNA synthetase (DARS2), which is released in circulation during bacterial pneumonia in humans and displays intrinsic innate immune properties and cellular repair properties. DARS2 interacts with a bacterial-induced ubiquitin E3 ligase subunit, FBXO24, which targets the synthetase for ubiquitylation and degradation, a process that is inhibited by DARS2 acetylation. During experimental pneumonia, Fbxo24 knockout mice exhibit elevated DARS2 levels with an increase in pulmonary cellular and cytokine levels. In silico modeling identified an FBXO24 inhibitory compound with immunostimulatory properties which extended DARS2 lifespan in cells. Here, we show a unique biological role for an extracellular, mitochondrially derived enzyme and its molecular control by the ubiquitin apparatus, which may serve as a mechanistic platform to enhance protective host immunity through small molecule discovery.

The mechanisms that guide T helper 2 (TH2) cell differentiation in barrier tissues are unclear. Here we describe the molecular pathways driving allergen-specific TH2 cells using temporal, spatial and single-cell transcriptomic tracking of house dust mite-specific T cells in mice. Differentiation and migration of lung allergen-specific TH2 cells requires early expression of the transcriptional repressor Blimp-1. Loss of Blimp-1 during priming in the lymph node ablated the formation of TH2 cells in the lung, indicating early Blimp-1 promotes TH2 cells with migratory capability. IL-2/STAT5 signals and autocrine/paracrine IL-10 from house dust mite-specific T cells were essential for Blimp-1 and subsequent GATA3 upregulation through repression of Bcl6 and Bach2. Spatial microniches of IL-2 in the lymph node supported the earliest Blimp-1+TH2 cells, demonstrating lymph node localization is a driver of TH2 initiation. Our findings identify an early requirement for IL-2-mediated spatial microniches that integrate with allergen-driven IL-10 from responding T cells to drive allergic asthma.

ABSTRACTBackgroundThe airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood.ObjectiveWe hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation.MethodsEthmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining andex vivofunctional analysis of sinus EpCs were used to validate our findings.ResultsAnalysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, andex vivoinhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma.ConclusionsTogether, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.KEY MESSAGESEpithelial mTORC1 activity is upregulated in CRSwNP.mTOR regulates EpC cytokine generation.Epithelial metabolic reprograming correlates with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in asthma.CAPSULE SUMMARYmTORC1 mediates EpC cytokine generation in CRSwNP.