Matthew J. Loza

The IL-12 Signature: NK Cell Terminal CD56+high Stage and Effector Functions

We report that human peripheral NK cells expressing high CD56 levels (CD56+high) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56+low NK cells. CD56+high NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56+) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56+high than on CD56+low NK cells. Consistent with their activated phenotype, many CD56+high NK cells are cycling and mediate heightened effector functions (proliferation, IFN-γ and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56+low NK cells all markers constitutively expressed on the CD56+high NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56−/+low NK cells acquire a CD56+high NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.

Assembly of Inflammation-Related Genes for Pathway-Focused Genetic Analysis

Recent identifications of associations between novel variants in inflammation-related genes and several common diseases emphasize the need for systematic evaluations of these genes in disease susceptibility. Considering that many genes are involved in the complex inflammation responses and many genetic variants in these genes have the potential to alter the functions and expression of these genes, we assembled a list of key inflammation-related genes to facilitate the identification of genetic associations of diseases with an inflammation-related etiology. We first reviewed various phases of inflammation responses, including the development of immune cells, sensing of danger, influx of cells to sites of insult, activation and functional responses of immune and non-immune cells, and resolution of the immune response. Assisted by the Ingenuity Pathway Analysis, we then identified 17 functional sub-pathways that are involved in one or multiple phases. This organization would greatly increase the chance of detecting gene-gene interactions by hierarchical clustering of genes with their functional closeness in a pathway. Finally, as an example application, we have developed tagging single nucleotide polymorphism (tSNP) arrays for populations of European and African descent to capture all the common variants of these key inflammation-related genes. Assays of these tSNPs have been designed and assembled into two Affymetrix ParAllele customized chips, one each for European (12,011 SNPs) and African (21,542 SNPs) populations. These tSNPs have greater coverage for these inflammation-related genes compared to the existing genome-wide arrays, particularly in the African population. These tSNP arrays can facilitate systematic evaluation of inflammation pathways in disease susceptibility. For additional applications, other genotyping platforms could also be employed. For existing genome-wide association data, this list of key inflammation-related genes and associated subpathways can facilitate comprehensive inflammation pathway- focused association analyses.

NKT and T cells: coordinate regulation of NK‐like phenotype and cytokine production

A maturation‐dependent change in phenotype and cytokine production from relatively immature CD161– or CD161+, IL‐13+IL‐4+, IFN‐γ–, to mature CD161+CD56+ IFN‐γ+ cells occurs in primary human α‐galactosyl ceramide‐reactive CD1d‐restricted natural killer T (NKT) cells under the control of IL‐12 and other monokines. Modulation of this process upon α‐galactosyl ceramide stimulation explains the opposite roles of NKT cells to drive type 1 and type 2 immune responses. Because the samedevelopmental changes occurred and were similarly regulated in T cells, the data establish that NKT cells should no longer be considered a functionally unique regulatory subset. However, the results of their analysis can be taken as a model for immunotherapeutic approaches with T cells for which a nominal or surrogate antigen is defined.

T-helper cell type 2 (Th2) and non-Th2 molecular phenotypes of asthma using sputum transcriptomics in U-BIOPRED.

Asthma is characterised by heterogeneous clinical phenotypes. Our objective was to determine molecular phenotypes of asthma by analysing sputum cell transcriptomics from 104 moderate-to-severe asthmatic subjects and 16 nonasthmatic subjects. After filtering on the differentially expressed genes between eosinophil- and noneosinophil-associated sputum inflammation, we used unbiased hierarchical clustering on 508 differentially expressed genes and gene set variation analysis of specific gene sets. We defined three transcriptome-associated clusters (TACs): TAC1 (characterised by immune receptors IL33R, CCR3 and TSLPR), TAC2 (characterised by interferon-, tumour necrosis factor-α- and inflammasome-associated genes) and TAC3 (characterised by genes of metabolic pathways, ubiquitination and mitochondrial function). TAC1 showed the highest enrichment of gene signatures for interleukin-13/T-helper cell type 2 (Th2) and innate lymphoid cell type 2. TAC1 had the highest sputum eosinophilia and exhaled nitric oxide fraction, and was restricted to severe asthma with oral corticosteroid dependency, frequent exacerbations and severe airflow obstruction. TAC2 showed the highest sputum neutrophilia, serum C-reactive protein levels and prevalence of eczema. TAC3 had normal to moderately high sputum eosinophils and better preserved forced expiratory volume in 1 s. Gene–protein coexpression networks from TAC1 and TAC2 extended this molecular classification. We defined one Th2-high eosinophilic phenotype TAC1, and two non-Th2 phenotypes TAC2 and TAC3, characterised by inflammasome-associated and metabolic/mitochondrial pathways, respectively. Clustering of transcriptomic genes from sputum cells defined one Th2- and two non-Th2-associated phenotypes http://ow.ly/UEkA3069ZYL

Distinction between IL‐13+ and IFN‐γ+ natural killer cells and regulation of their pool size by IL‐4

The hypothesis that distinct subsets of NK cells produce type 2 and type 1 cytokines in resting naive lymphocytes was tested analyzing cytokine production at the single‐cell level. Two non‐overlapping IL‐13+ and IFN‐γ+ subsets were identified in adult and neonatal NK cells. IL‐2 maintained their relative proportion. Accumulation of the former was induced by IL‐4, but not IL‐13, and inhibited by IL‐12; that of the latter was induced by IL‐12 and inhibited by IL‐4 and IL‐13. IL‐4 induced preferential proliferation of the pre‐existing peripheral IL‐13+ cells, whereas IL‐12 had minimal effect on proliferation of the IFN‐γ+ NK cells. The IL‐13+ cells (CD161+ only) are phenotypically distinct from the IFN‐γ+ones (CD56+) before and after culture under any condition analyzed, and produce IL‐13 in response to NK‐sensitive target cells and PMA + Ca2+ ionophore, whereas also FcγRIIIA and IL‐2 + IL‐12 stimulate IFN‐γ production. These data define the existence and regulation of two distinct resting peripheral NK cell subsets producing type 1 and type 2 cytokines, and suggest possible roles for IL‐13+ NK cells in allergy.

A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED

Rationale: Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. Objectives: Using transcriptomic profiling of airway tissues, we sought to define the molecular phenotypes of severe asthma. Methods: The transcriptome derived from bronchial biopsies and epithelial brushings of 107 subjects with moderate to severe asthma were annotated by gene set variation analysis using 42 gene signatures relevant to asthma, inflammation, and immune function. Topological data analysis of clinical and histologic data was performed to derive clusters, and the nearest shrunken centroid algorithm was used for signature refinement. Measurements and Main Results: Nine gene set variation analysis signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T‐helper cell type 2 cytokines and lack of corticosteroid response (group 1 and group 3). Group 1 had the highest submucosal eosinophils, as well as high fractional exhaled nitric oxide levels, exacerbation rates, and oral corticosteroid use, whereas group 3 patients showed the highest levels of sputum eosinophils and had a high body mass index. In contrast, group 2 and group 4 patients had an 86% and 64% probability, respectively, of having noneosinophilic inflammation. Using machine learning tools, we describe an inference scheme using the currently available inflammatory biomarkers sputum eosinophilia and fractional exhaled nitric oxide levels, along with oral corticosteroid use, that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. Conclusions: This analysis demonstrates the usefulness of a transcriptomics‐driven approach to phenotyping that segments patients who may benefit the most from specific agents that target T‐helper cell type 2‐mediated inflammation and/or corticosteroid insensitivity.

Glucocorticoid- and Protein Kinase A―Dependent Transcriptome Regulation in Airway Smooth Muscle

Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.

Linear ‘2–0–1’ lymphocyte development: hypotheses on cellular bases for immunity

Abstract It has been proposed that immune responses to intra- (type 1) and extra-cellular (type 2) pathogens are regulated by two T-cell subsets branching from type 0 cells on T-cell receptor (TCR) engagement and terminally differentiating in distinct cytokine environments. However, analysis at the single-cell level of human T cells and natural killer (NK) cells has revealed that peripheral immature cells of both lineages exist, produce only type 2 cytokines and either proliferate or differentiate to interferon-γ producing cells in distinct cytokine environments, even without TCR engagement. These data support the hypothesis that a modified balance between proliferation and/or survival and differentiation of immature type 2 cytokine-producing cells regulates productive, type 1, immune responses. This new concept provides a simpler and testable framework to understand and manipulate the immune system and immune pathologies.

Inflammatory Profile and Response to Anti-Tumor Necrosis Factor Therapy in Patients with Chronic Pulmonary Sarcoidosis†

ABSTRACT Sarcoidosis is an inflammatory, granulomatous disease of unknown etiology that most commonly afflicts the lungs. Despite aggressive immunosuppressive therapies, many sarcoidosis patients still chronically present significant symptoms. Infliximab, a therapeutic tumor necrosis factor alpha (TNF-α) monoclonal antibody (MAb), produced a small but significant improvement in forced vital capacity (FVC) in sarcoidosis patients in a double-blind, placebo-controlled, phase II clinical trial. In the current study, serum samples from this clinical trial were assessed to evaluate the underlying hypothesis that treatment with infliximab would reduce systemic inflammation associated with sarcoidosis, correlating with the extent of clinical response. A 92-analyte multiplex panel was used to assess the expression of serum proteins in 134 sarcoidosis patients compared with sera from 50 healthy controls. A strong systemic inflammatory profile was associated with sarcoidosis, with 29 analytes significantly elevated in sarcoidosis (false-discovery rate, <0.05 and >50% higher than controls). The associated analytes included chemokines, neutrophil-associated proteins, acute-phase proteins, and metabolism-associated proteins. This profile was evident despite patients receiving corticosteroids and immunosuppressive therapies. Following infliximab treatment, sarcoidosis patients expressing the highest levels of TNF-α, who had more severe disease, had the greatest improvement in FVC and reduction in serum levels of the inflammatory proteins MIP-1β and TNF-RII. This study supports the need for further exploration of anti-TNF therapy for chronic sarcoidosis patients, particularly for those expressing the highest serum levels of TNF-α.

Validated and longitudinally stable asthma phenotypes based on cluster analysis of the ADEPT study.

BackgroundAsthma is a disease of varying severity and differing disease mechanisms. To date, studies aimed at stratifying asthma into clinically useful phenotypes have produced a number of phenotypes that have yet to be assessed for stability and to be validated in independent cohorts. The aim of this study was to define and validate, for the first time ever, clinically driven asthma phenotypes using two independent, severe asthma cohorts: ADEPT and U-BIOPRED.MethodsFuzzy partition-around-medoid clustering was performed on pre-specified data from the ADEPT participants (n = 156) and independently on data from a subset of U-BIOPRED asthma participants (n = 82) for whom the same variables were available. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and to a larger subset of the U-BIOPRED asthma dataset (n = 397). High and low type-2 inflammation phenotypes were defined as high or low Th2 activity, indicated by endobronchial biopsies gene expression changes downstream of IL-4 or IL-13.ResultsFour phenotypes were identified in the ADEPT (training) cohort, with distinct clinical and biomarker profiles. Phenotype 1 was “mild, good lung function, early onset”, with a low-inflammatory, predominantly Type-2, phenotype. Phenotype 2 had a “moderate, hyper-responsive, eosinophilic” phenotype, with moderate asthma control, mild airflow obstruction and predominant Type-2 inflammation. Phenotype 3 had a “mixed severity, predominantly fixed obstructive, non-eosinophilic and neutrophilic” phenotype, with moderate asthma control and low Type-2 inflammation. Phenotype 4 had a “severe uncontrolled, severe reversible obstruction, mixed granulocytic” phenotype, with moderate Type-2 inflammation. These phenotypes had good longitudinal stability in the ADEPT cohort. They were reproduced and demonstrated high classification probability in two subsets of the U-BIOPRED asthma cohort.ConclusionsFocusing on the biology of the four clinical independently-validated easy-to-assess ADEPT asthma phenotypes will help understanding the unmet need and will aid in developing tailored therapies.Trial registrationNCT01274507 (ADEPT), registered October 28, 2010 and NCT01982162 (U-BIOPRED), registered October 30, 2013.