ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1.

Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.

T cell-dependent bispecific antibodies alter organ-specific endothelial cell-T cell interaction.

Preclinical and clinical studies demonstrate that T cell-dependent bispecific antibodies (TDBs) induce systemic changes in addition to tumor killing, leading to adverse events. Here, we report an in-depth characterization of acute responses to TDBs in tumor-bearing mice. Contrary to modest changes in tumors, rapid and substantial lymphocyte accumulation and endothelial cell (EC) activation occur around large blood vessels in normal organs including the liver. We hypothesize that organ-specific ECs may account for the differential responses in normal tissues and tumors, and we identify a list of genes selectively upregulated by TDB in large liver vessels. Using one of the genes as an example, we demonstrate that CD9 facilitates ICAM-1 to support T cell-EC interaction in response to soluble factors released from a TDB-mediated cytotoxic reaction. Our results suggest that multiple factors may cooperatively promote T cell infiltration into normal organs as a secondary response to TDB-mediated tumor killing. These data shed light on how different vascular beds respond to cancer immunotherapy and may help improve their safety and efficacy.

Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α.

Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.

ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses.

Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.

Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach.

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay's specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay's specificity and build confidence in a study's conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study's results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.

A randomized, placebo-controlled trial evaluating effects of lebrikizumab on airway eosinophilic inflammation and remodelling in uncontrolled asthma (CLAVIER).

BACKGROUND: The anti-interleukin 13 (IL-13) monoclonal antibody lebrikizumab improves lung function in patients with moderate-to-severe uncontrolled asthma, but its effects on airway inflammation and remodelling are unknown. CLAVIER was designed to assess lebrikizumab's effect on eosinophilic inflammation and remodelling. OBJECTIVE: To report safety and efficacy results from enrolled participants with available data from CLAVIER. METHODS: We performed bronchoscopy on patients with uncontrolled asthma before and after 12 weeks of randomized double-blinded treatment with lebrikizumab (n = 31) or placebo (n = 33). The pre-specified primary end-point was relative change in airway subepithelial eosinophils per mm2 of basement membrane (cells/mm2 ). Pre-specified secondary and exploratory outcomes included change in IL-13-associated biomarkers and measures of airway remodelling. RESULTS: There was a baseline imbalance in tissue eosinophils and high variability between treatment groups. There was no discernible change in adjusted mean subepithelial eosinophils/mm2 in response to lebrikizumab (95% CI, -82.5%, 97.5%). As previously observed, FEV1 increased after lebrikizumab treatment. Moreover, subepithelial collagen thickness decreased 21.5% after lebrikizumab treatment (95% CI, -32.9%, -10.2%), and fractional exhaled nitric oxide, CCL26 and SERPINB2 mRNA expression in bronchial tissues also reduced. Lebrikizumab was well tolerated, with a safety profile consistent with other lebrikizumab asthma studies. CONCLUSIONS & CLINICAL RELEVANCE: We did not observe reduced tissue eosinophil numbers in association with lebrikizumab treatment. However, in pre-specified exploratory analyses, lebrikizumab treatment was associated with reduced degree of subepithelial fibrosis, a feature of airway remodelling, as well as improved lung function and reduced key pharmacodynamic biomarkers in bronchial tissues. These results reinforce the importance of IL-13 in airway pathobiology and suggest that neutralization of IL-13 may reduce asthmatic airway remodelling. CLINICAL TRIAL REGISTRATION: NCT02099656.

IRE1α Disruption in Triple-Negative Breast Cancer Cooperates with Antiangiogenic Therapy by Reversing ER Stress Adaptation and Remodeling the Tumor Microenvironment.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Detection of Necroptosis by Phospho-RIPK3 Immunohistochemical Labeling.

Activation of the kinase RIPK3 (receptor interacting protein kinase 3) is a hallmark of cells dying by necroptosis. RIPK3 phosphorylates both itself and the pseudokinase MLKL (mixed lineage kinase-like) resulting in MLKL translocation to membranes and cell lysis. Antibodies recognizing RIPK3 autophosphorylation or the RIPK3-dependent phosphorylation sites on MLKL have therefore been used to monitor necroptosis induction. Here we describe immunohistochemical labeling for autophosphorylated mouse RIPK3 as a means of detecting cells undergoing necroptosis in mouse tissues.

Correction: Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers.

Notch ligands signal through one of four receptors on neighboring cells to mediate cell-cell communication and control cell fate, proliferation, and survival. Although aberrant Notch activation has been implicated in numerous malignancies, including breast cancer, the importance of individual receptors in distinct breast cancer subtypes and the mechanisms of receptor activation remain unclear. Using a novel antibody to detect active NOTCH3, we report here that NOTCH3 signals constitutively in a panel of basal breast cancer cell lines and in more than one third of basal tumors. Selective inhibition of individual ligands revealed that this signal does not require canonical ligand induction. A NOTCH3 antagonist antibody inhibited growth of basal lines, whereas a NOTCH3 agonist antibody enhanced the transformed phenotype in vitro and in tumor xenografts. Transcriptomic analyses generated a Notch gene signature that included Notch pathway components, the oncogene c-Myc, and the mammary stem cell regulator Id4 This signature drove clustering of breast cancer cell lines and tumors into the common subtypes and correlated with the basal classification. Our results highlight an unexpected ligand-independent induction mechanism and suggest that constitutive NOTCH3 signaling can drive an oncogenic program in a subset of basal breast cancers. Cancer Res; 77(6); 1439-52. ©2017 AACR.

RIPK1 inhibits ZBP1-driven necroptosis during development.

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Depletion of major pathogenic cells in asthma by targeting CRTh2.

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.

Preclinical Safety Profile of a Depleting Antibody against CRTh2 for Asthma: Well Tolerated Despite Unexpected CRTh2 Expression on Vascular Pericytes in the Central Nervous System and Gastric Mucosa.

CRTh2 is expressed on immune cells that drive asthma pathophysiology. Current treatment options for severe asthma are inadequate and therapeutic antibody-mediated depletion of CRTh2-expressing cells represents a promising new therapeutic strategy. Here we report for the first time that CRTh2 is not only expressed on immune cells, but also on microvasculature in the central nervous system (CNS) and gastric mucosa in humans. Microvascular expression of CRTh2 raises a safety concern because a therapeutic antiCRTh2 antibody with enhanced depletion capacity could lead to vascular damage. To evaluate this safety risk, we characterized microvascular expression in human and in transgenic mice expressing human CRTh2 protein (hCRTh2.BAC.Tg) and found that CRTh2 is not localized to microvascular endothelium that is directly exposed to circulating therapeutic antibody, but rather, to pericytes that in the CNS are shielded from direct circulatory exposure by the blood-brain barrier. Immunohistochemical visualization of an intravenously administered antiCRTh2 antibody in transgenic mice revealed localization to microvascular pericytes in the gastric mucosa but not in the CNS, suggesting the blood-brain barrier effectively limits pericyte exposure to circulating therapeutic antibody in the CNS. Repeated dosing with a depleting antiCRTh2 antibody in hCRTh2.BAC.Tg mice revealed linear pharmacokinetics and no drug-related adverse findings in any tissues, including the CNS and gastric mucosa, despite complete depletion of CRTh2 expressing circulating eosinophils and basophils. Collectively, these studies demonstrate that the likelihood of drug-related CNS or gastrointestinal toxicity in humans treated with a therapeutic depleting antiCRTh2 antibody is low despite pericyte expression of CRTh2 in these tissues.

Effect of selective LRRK2 kinase inhibition on nonhuman primate lung.

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

Lactate dehydrogenase B is required for the growth of KRAS-dependent lung adenocarcinomas.

PURPOSE: This study is aimed to identify genes within the KRAS genomic amplicon that are both coupregulated and essential for cell proliferation when KRAS is amplified in lung cancer. EXPERIMENTAL DESIGN: We used an integrated genomic approach to identify genes that are coamplified with KRAS in lung adenocarcinomas and subsequently preformed an RNA interference (RNAi) screen to uncover functionally relevant genes. The role of lactate dehydrogenase B (LDHB) was subsequently investigated both in vitro and in vivo by siRNA and short hairpin RNA (shRNA)-mediated knockdown in a panel of lung adenocarcinoma cells lines. LDHB expression was also investigated in patient tumors using microarray and immunohistochemistry analyses. RESULTS: RNAi-mediated depletion of LDHB abrogated cell proliferation both in vitro and in xenografted tumors in vivo. We find that LDHB expression correlates to both KRAS genomic copy number gain and KRAS mutation in lung cancer cell lines and adenocarcinomas. This correlation between LDHB expression and KRAS status is specific for lung cancers and not other tumor types that harbor KRAS mutations. Consistent with a role for LDHB in glycolysis and tumor metabolism, KRAS-mutant lung tumors exhibit elevated expression of a glycolysis gene signature and are more dependent on glycolysis for proliferation compared with KRAS wild-type lung tumors. Finally, high LDHB expression was a significant predictor of shorter survival in patients with lung adenocarcinomas. CONCLUSION: This study identifies LDHB as a regulator of cell proliferation in a subset of lung adenocarcinoma and may provide a novel therapeutic approach for treating lung cancer.

Airway epithelial miRNA expression is altered in asthma.

RATIONALE: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. OBJECTIVES: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. METHODS: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. MEASUREMENTS AND MAIN RESULTS: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. CONCLUSIONS: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).

The H antigen at epithelial surfaces is associated with susceptibility to asthma exacerbation.

RATIONALE: Acute asthma exacerbations, precipitated by viral infections, are a significant cause of morbidity, but not all patients with asthma are equally susceptible. OBJECTIVES: To explore susceptibility factors for asthma exacerbations, we considered a role for histoblood group antigens because they are implicated in mechanisms of gastrointestinal viral infection, specifically the O-secretor mucin glycan phenotype. We investigated if this phenotype is associated with susceptibility to asthma exacerbation. METHODS: We performed two consecutive case-control studies in subjects with asthma who were either prone or resistant to asthma exacerbations. Exacerbation-prone cases had frequent use of prednisone for an asthma exacerbation and frequent asthma-related healthcare utilization, whereas exacerbation-resistant control subjects had rarely reported asthma exacerbations. The frequency of different mucin glycan phenotypes, defined by the presence or absence of H (O), A, B, or AB antigens, was compared in cases and control subjects. MEASUREMENTS AND MAIN RESULTS: In an initial study consisting of 49 subjects with asthma (23 cases and 26 control subjects), we found that having the O-secretor phenotype was associated with a 5.8-fold increase in the odds of being a case (95% confidence interval, 1.7-21.0; P = 0.006). In a replication study consisting of 204 subjects with asthma (101 cases and 103 control subjects), we found that having the O-secretor phenotype was associated with a 2.3-fold increased odds of being a case (95% confidence interval, 1.2-4.4; P = 0.02). CONCLUSIONS: The O-secretor mucin glycan phenotype is associated with susceptibility to asthma exacerbation. Clinical trial registered at www.clinicaltrials.gov (NCT00201266).