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Exogenous electric fields are currently used in human therapy in a number of contexts. Interestingly, electric fields have also been shown to alter migration and function of immune cells, suggesting the potential for electric field-based immune therapy. Little is known as to the effect of electric field treatment (EFT) on the lung. To determine if EFT associates with changes in lung immune cell infiltration, we used a mouse model with varying methods of EFT application and measured cells and soluble mediators using flow cytometry and cytokine/chemokine multiplex. EFT was associated with a transient increase in lung neutrophils and decrease in eosinophils in naïve mice within 2 h of treatment, accompanied by an increase in IL-6 levels. In order to test whether EFT could alter eosinophil/neutrophil recruitment in a relevant disease model, a mouse model of allergic airway inflammation was used. Four EFT doses in allergen-sensitized mice resulted in increased neutrophil and reduced eosinophil infiltrates following allergen challenge, suggesting a durable change in inflammation by EFT. Mice with allergic inflammation were analyzed by flexiVent for measures of lung function. EFT-treated mice had increased inspiratory capacity and other measures of lung function were not diminished. These data suggest EFT may be used to manipulate immune cell infiltration in the lung without affecting lung function.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.

Lysophosphatidic acid (LPA) is a pleiotropic lipid signaling molecule associated with asthma pathobiology. LPA elicits its effects by binding to at least six known cell surface G protein-coupled receptors (LPA1-6) that are expressed in the lung in a cell type-specific manner. LPA2 in particular has emerged as an attractive therapeutic target in asthma because it appears to transduce inhibitory or cell-protective signals. We studied a novel and specific small molecule LPA2 agonist (2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl] benzoic acid [DBIBB]) in a mouse model of house dust mite-induced allergic airway inflammation. Mice injected with DBIBB developed significantly less airway and lung inflammation compared with vehicle-treated controls. Levels of lung Th2 cytokines were also significantly attenuated by DBIBB. We conclude that pharmacologic activation of LPA2 attenuates Th2-driven allergic airway inflammation in a mouse model of asthma. Targeting LPA receptor signaling holds therapeutic promise in allergic asthma.

Recent published studies have highlighted the complexity of the immune response to allergens, and the various asthma phenotypes that arise as a result. Although the interplay of regulatory and effector immune cells responding to allergen would seem to dictate the nature of the asthmatic response, little is known regarding how tolerance versus reactivity to allergen occurs in the lung. The vast majority of mouse models study allergen encounter in naive animals, and therefore exclude the possibility that previous encounters with allergen may influence future sensitization. To address this, we studied sensitization to the model allergen OVA in mice in the context of pre-existing tolerance to OVA. Allergen sensitization by either systemic administration of OVA with aluminum hydroxide or mucosal administration of OVA with low-dose LPS was suppressed in tolerized animals. However, higher doses of LPS induced a mixed Th2 and Th17 response to OVA in both naive and tolerized mice. Of interest, tolerized mice had more pronounced Th17-type inflammation than did naive mice receiving the same sensitization, suggesting pre-existing tolerance altered the inflammatory phenotype. These data show that a pre-existing tolerogenic immune response to allergen can affect subsequent sensitization in the lung. These findings have potential significance for understanding late-onset disease in individuals with severe asthma.

Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2(-/-) mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB-driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2(-/-) DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2(-/-) mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.

Asthma is a heterogeneous disease, with multiple underlying inflammatory pathways and structural airway abnormalities that impact disease persistence and severity. Recent progress has been made in developing targeted asthma therapeutics, especially for subjects with eosinophilic asthma. However, there is an unmet need for new approaches to treat patients with severe and exacerbation-prone asthma, who contribute disproportionately to disease burden. Extensive deep phenotyping has revealed the heterogeneous nature of severe asthma and identified distinct disease subtypes. A current challenge in the field is to translate new and emerging knowledge about different pathobiologic mechanisms in asthma into patient-specific therapies, with the ultimate goal of modifying the natural history of disease. Here, we describe the Precision Interventions for Severe and/or Exacerbation-Prone Asthma (PrecISE) Network, a groundbreaking collaborative effort of asthma researchers and biostatisticians from around the United States. The PrecISE Network was designed to conduct phase II/proof-of-concept clinical trials of precision interventions in the population with severe asthma, and is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health. Using an innovative adaptive platform trial design, the PrecISE Network will evaluate up to 6 interventions simultaneously in biomarker-defined subgroups of subjects. We review the development and organizational structure of the PrecISE Network, and choice of interventions being studied. We hope that the PrecISE Network will enhance our understanding of asthma subtypes and accelerate the development of therapeutics for severe asthma.

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design.

The Precision Interventions for Severe and/or Exacerbation-prone Asthma (PrecISE) study is an adaptive platform trial designed to investigate novel interventions to severe asthma. The study is conducted under a master protocol and utilizes a crossover design with each participant receiving up to five interventions and at least one placebo. Treatment assignments are based on the patients' biomarker profiles and precision health methods are incorporated into the interim and final analyses. We describe key elements of the PrecISE study including the multistage adaptive enrichment strategy, early stopping of an intervention for futility, power calculations, and the primary analysis strategy.

Asthma is a common chronic disease without cure. Our understanding of asthma onset, pathobiology, classification, and management has evolved substantially over the past decade; however, significant asthma-related morbidity and excess healthcare use and costs persist. To address this important clinical condition, the NHLBI convened a group of extramural investigators for an Asthma Research Strategic Planning workshop on September 18-19, 2014, to accelerate discoveries and their translation to patients. The workshop focused on (1) in utero and early-life origins of asthma, (2) the use of phenotypes and endotypes to classify disease, (3) defining disease modification, (4) disease management, and (5) implementation research. This report summarizes the workshop and produces recommendations to guide future research in asthma.

Abstract: The finding that murine and simian cells have differential susceptibility to diphtheria toxin (DTx) led to the development of genetically engineered mouse strains that express the simian or human diphtheria toxin receptor (DTR) under the control of various mouse gene promoters. Injection of DTx into DTR engineered mice allows for rapid and transient depletion of various cell populations. There are several advantages to this approach over global knockout mice, including normal mouse development and temporal control over when cell depletion occurs. As a result, many DTR engineered mouse strains have been developed, resulting in significant insights into the cell biology of various disease states. We used Foxp3DTR mice to attempt local depletion of Foxp3+ cells in the lung in a model of tolerance breakdown. Intratracheal administration of DTx resulted in robust depletion of lung Foxp3+ cells. However, DTx administration was accompanied by significant local inflammation, even in control C57Bl/6 mice. These data suggest that DTx administration to non-transgenic mice is not always an immunologically inert event, and proper controls must be used to assess various DTx-mediated depletion regimens. [Copyright &y& Elsevier]