Risankizumab in Severe Asthma - A Phase 2a, Placebo-Controlled Trial.

BACKGROUND: Interleukin-23 has been implicated in airway inflammation that is mediated by type 2 and type 17 cytokines. Whether targeting interleukin-23 in the treatment of asthma improves disease control and reduces airway inflammation is unclear. METHODS: We conducted a phase 2a, multicenter, randomized, double-blind, placebo-controlled, 24-week, parallel-group trial to assess the efficacy and safety of risankizumab, an anti-interleukin-23p19 monoclonal antibody, in adults with severe asthma. Patients were assigned to receive 90 mg of risankizumab or placebo, administered subcutaneously once every 4 weeks. The primary end point was the time to the first asthma worsening. Asthma worsening was defined as deterioration from baseline on 2 or more consecutive days; deterioration was considered to be a decrease of at least 30% in the morning peak expiratory flow or an increase from baseline of at least 50% in the number of puffs of rescue medication in a 24-hour period (equating to at least four additional puffs), a severe asthma exacerbation, or an increase of 0.75 or more points on the 5-item Asthma Control Questionnaire (ACQ-5; scores range from 0 to 6, with higher scores indicating less control). Secondary end points were the annualized rate of asthma worsening, the annualized rate of severe exacerbations, the ACQ-5 score, and the forced expiratory volume in 1 second. Exploratory end points were assessed with the use of sputum cytologic analysis and gene expression analysis, and safety was assessed. RESULTS: A total of 105 patients received risankizumab and 109 received placebo. The clinical characteristics of the patients were similar in the two groups. The time to the first asthma worsening was shorter with risankizumab than with placebo (median, 40 days vs. 86 days; hazard ratio, 1.46; 95% confidence interval [CI], 1.05 to 2.04; P = 0.03). The rate ratio for annualized asthma worsening with risankizumab as compared with placebo was 1.49 (95% CI, 1.12 to 1.99), and the rate ratio for severe exacerbations was 1.13 (95% CI, 0.75 to 1.70). Sputum transcriptomic pathway analysis showed that genes involved in the activation of natural killer cells and cytotoxic T cells and the activation of the type 1 helper T and type 17 helper T transcription factors were down-regulated by risankizumab. No safety concerns were associated with risankizumab therapy. CONCLUSIONS: Risankizumab treatment was not beneficial in severe asthma. The time to the first asthma worsening was shorter and the annualized rate of asthma worsening was higher with risankizumab than with placebo. (Funded by AbbVie and Boehringer Ingelheim; ClinicalTrials.gov number, NCT02443298.).

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo: A Clinical Trial.

Rationale: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. Objectives: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P < 0.05). In healthy subjects, ILC1s increased from baseline at Day 3 (P = 0.001), while in patients with asthma, ILC1s increased from baseline at Day 8 (P = 0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P = 0.024) and Day 8 (P = 0.005). Increased ILC2:ILC1 ratio in patients with asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions: An ILC2-predominant inflammatory profile in patients with asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations.

IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent.

Cutaneous group 2 innate lymphoid cells (ILC2) are spatially and epigenetically poised to respond to barrier compromise and associated immunological threats. ILC2, lacking rearranged antigen-specific receptors, are primarily activated by damage-associated cytokines and respond with type 2 cytokine production. To investigate ILC2 potential for direct sensing of skin pathogens and allergens, we performed RNA sequencing of ILC2 derived from in vivo challenged human skin or blood. We detected expression of NOD2 and TLR2 by skin and blood ILC2. Stimulation of ILC2 with TLR2 agonist alone not only induced interleukin-5 (IL-5) and IL-13 expression but also elicited IL-6 expression in combination with Staphylococcus aureus muramyl dipeptide (MDP). Heat-killed skin-resident bacteria provoked an IL-6 profile in ILC2 in vitro that was notably impaired in ILC2 derived from patients with nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations. In addition, we show that NOD2 signaling can stimulate autophagy in ILC2, which was also impaired in patients with NOD2 mutations. Here, we have identified a role for ILC2 NOD2 signaling in the differential regulation of ILC2-derived IL-6 and have reported a previously unrecognized pathway of direct ILC2 bacterial sensing.

Profiling of H3K27Ac Reveals the Influence of Asthma on the Epigenome of the Airway Epithelium.

BACKGROUND: Asthma is a chronic airway disease driven by complex genetic-environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood. METHODS: We piloted genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma (n = 4) and healthy controls (n = 3). RESULTS: We identified n = 4,321 (FDR < 0.05) regions exhibiting differential H3K27ac enrichment between asthma and health, clustering at genes associated predominately with epithelial processes (EMT). We identified initial evidence of asthma-associated Super-Enhancers encompassing genes encoding transcription factors (TP63) and enzymes regulating lipid metabolism (PTGS1). We integrated published datasets to identify epithelium-specific transcription factors associated with H3K27ac in asthma (TP73) and identify initial relationships between asthma-associated changes in H3K27ac and transcriptional profiles. Finally, we investigated the potential of CRISPR-based approaches to functionally evaluate H3K27ac-asthma landscape in vitro by identifying guide-RNAs capable of targeting acetylation to asthma DERs and inducing gene expression (TLR3). CONCLUSION: Our small pilot study validates genome-wide approaches for deciphering epigenetic mechanisms underlying asthma pathogenesis in the airways.

Human retinoic acid-regulated CD161+ regulatory T cells support wound repair in intestinal mucosa.

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn's disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn's disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.

Effects of vitamin D on inflammatory and oxidative stress responses of human bronchial epithelial cells exposed to particulate matter.

BACKGROUND: Particulate matter (PM) pollutant exposure, which induces oxidative stress and inflammation, and vitamin D insufficiency, which compromises immune regulation, are detrimental in asthma. OBJECTIVES: Mechanistic cell culture experiments were undertaken to ascertain whether vitamin D abrogates PM-induced inflammatory responses of human bronchial epithelial cells (HBECs) through enhancement of antioxidant pathways. METHODS: Transcriptome analysis, PCR and ELISA were undertaken to delineate markers of inflammation and oxidative stress; with comparison of expression in primary HBECs from healthy and asthmatic donors cultured with reference urban PM in the presence/absence of vitamin D. RESULTS: Transcriptome analysis identified over 500 genes significantly perturbed by PM-stimulation, including multiple pro-inflammatory cytokines. Vitamin D altered expression of a subset of these PM-induced genes, including suppressing IL6. Addition of vitamin D suppressed PM-stimulated IL-6 production, although to significantly greater extent in healthy versus asthmatic donor cultures. Vitamin D also differentially affected PM-stimulated GM-CSF, with suppression in healthy HBECs and enhancement in asthmatic cultures. Vitamin D increased HBEC expression of the antioxidant pathway gene G6PD, increased the ratio of reduced to oxidised glutathione, and in PM-stimulated cultures decreased the formation of 8-isoprostane. Pre-treatment with vitamin D decreased CXCL8 and further decreased IL-6 production in PM-stimulated cultures, an effect abrogated by inhibition of G6PD with DHEA, supporting a role for this pathway in the anti-inflammatory actions of vitamin D. CONCLUSIONS: In a study using HBECs from 18 donors, vitamin D enhanced HBEC antioxidant responses and modulated the immune response to PM, suggesting that vitamin D may protect the airways from pathological pollution-induced inflammation.

miR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells.

Class-switch recombination (CSR) is an essential B cell process that alters the isotype of antibody produced by the B cell, tailoring the immune response to the nature of the invading pathogen. CSR requires the activity of the mutagenic enzyme AID (encoded by AICDA) to generate chromosomal lesions within the immunoglobulin genes that initiate the class switching recombination event. These AID-mediated mutations also participate in somatic-hypermutation of the immunoglobulin variable region, driving affinity maturation. As such, AID poses a significant oncogenic threat if it functions outside of the immunoglobulin locus. We found that expression of the microRNA, miR-29b, was repressed in B cells isolated from tonsil tissue, relative to circulating naïve B cells. Further investigation revealed that miR-29b was able to directly initiate the degradation of AID mRNA. Enforced overexpression of miR-29b in human B cells precipitated a reduction in overall AID protein and a corresponding diminution in CSR to IgE. Given miR-29b's ability to potently target AID, a mutagenic molecule that can initiate chromosomal translocations and "off-target" mutations, we propose that miR-29b acts to silence premature AID expression in naïve B cells, thus reducing the likelihood of inappropriate and potentially dangerous deamination activity.

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention.

Pinning allergies on pathogenic TH2 cells.

A new proinflammatory subtype of antigen-specific TH2 cell that expresses CD161 emerges as the pathogenic cell type in allergic disease and is deleted during allergen-specific immunotherapy (Wambre et al, this issue).

Mucosal Type 2 Innate Lymphoid Cells Are a Key Component of the Allergic Response to Aeroallergens.

RATIONALE: Newly characterized type 2 innate lymphoid cells (ILC2s) display potent type 2 effector functionality; however, their contribution to allergic airways inflammation and asthma is poorly understood. Mucosal biopsy used to characterize the airway mucosa is invasive, poorly tolerated, and does not allow for sequential sampling. OBJECTIVES: To assess the role of ILC2s during nasal allergen challenge in subjects with allergic rhinitis using novel noninvasive methodology. METHODS: We used a human experimental allergen challenge model, with flow cytometric analysis of nasal curettage samples, to assess the recruitment of ILC2s and granulocytes to the upper airways of subjects with atopy and healthy subjects after allergen provocation. Soluble mediators in the nasal lining fluid were measured using nasosorption. MEASUREMENTS AND MAIN RESULTS: After an allergen challenge, subjects with atopy displayed rapid induction of upper airway symptoms, an enrichment of ILC2s, eosinophils, and neutrophils, along with increased production of IL-5, prostaglandin D2, and eosinophil and T-helper type 2 cell chemokines compared with healthy subjects. The most pronounced ILC2 recruitment was observed in subjects with elevated serum IgE and airway eosinophilia. CONCLUSIONS: The rapid recruitment of ILC2s to the upper airways of allergic patients with rhinitis, and their association with key type 2 mediators, highlights their likely important role in the early allergic response to aeroallergens in the airways. The novel methodology described herein enables the analysis of rare cell populations from noninvasive serial tissue sampling.

Intradermal grass pollen immunotherapy increases TH2 and IgE responses and worsens respiratory allergic symptoms.

BACKGROUND: Repeated low-dose grass pollen intradermal allergen injection suppresses allergen-induced cutaneous late-phase responses comparably with conventional subcutaneous and sublingual immunotherapy. OBJECTIVE: We sought to evaluate the efficacy and safety of grass pollen intradermal immunotherapy in the treatment of allergic rhinitis. METHODS: We randomly assigned 93 adults with grass pollen-induced allergic rhinitis to receive 7 preseasonal intradermal allergen injections (containing 7 ng of Phl p 5 major allergen) or a histamine control. The primary end point was daily combined symptom-medication scores during the 2013 pollen season (area under the curve). Analysis was by intention to treat. Skin biopsy specimens were collected after intradermal allergen challenges, and late-phase responses were measured 4 and 7, 10, or 13 months after treatment. RESULTS: There was no significant difference in the primary end point between treatment arms (active, n = 46; control, n = 47; median difference, 14; 95% CI, -172.5 to 215.1; P = .80). Among secondary end points, nasal symptoms were worse in the intradermal treatment group, as measured based on daily (median difference, 35; 95% CI, 4.0-67.5; P = .03) and visual analog scale (median difference, 53; 95% CI, -11.6 to 125.2; P = .05) scores. In a per-protocol analysis intradermal immunotherapy was further associated with worse asthma symptoms and fewer symptom-free days. Intradermal immunotherapy increased serum Phleum pratense-specific IgE levels (P = .001) compared with those in the control arm. T cells cultured from biopsy specimens of subjects undergoing intradermal immunotherapy had higher expression of the TH2 surface marker CRTH2 (P = .04) and lower expression of the TH1 marker CXCR3 (P = .01), respectively. Late-phase responses remained inhibited 7 months after treatment (P = .03). CONCLUSION: Intradermal allergen immunotherapy suppressed skin late-phase responses but was not clinically effective and resulted in worsening of respiratory allergic symptoms.

T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates.

Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression.

Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.

Group 2 Innate Lymphoid Cells Express Functional NKp30 Receptor Inducing Type 2 Cytokine Production.

Group 2 innate lymphoid cells (ILC2) are important in effector functions for eliciting allergic inflammation, parasite defense, epithelial repair, and lipid homeostasis. ILC2 lack rearranged Ag-specific receptors, and although many soluble factors such as cytokines and lipid mediators can influence ILC2, direct interaction of these cells with the microenvironment and other cells has been less explored. Natural cytotoxicity receptors are expressed by subsets of group 1 ILC and group 3 ILC and thought to be important for their effector function, but they have not been shown to be expressed by ILC2. Therefore, we sought to investigate the expression and functional properties of the natural cytotoxicity receptor NKp30 on human ILC2. A subset of ex vivo and cultured ILC2 express NKp30 that upon interaction with its cognate activatory ligand B7-H6 induces rapid production of type 2 cytokines. This interaction can be blocked by NKp30 blocking Ab and an inhibitory ligand, galectin-3. Higher expression of B7-H6 was observed in lesional skin biopsies of patients with atopic dermatitis, and incubation of keratinocytes with proinflammatory and type 2 cytokines upregulated B7-H6, leading to increased ILC2 cytokine production. NKp30-B7-H6 interaction is a novel cell contact mechanism that mediates activation of ILC2 and identifies a potential target for the development of novel therapeutics for atopic dermatitis and other atopic diseases.

IL-25/IL-33-responsive TH2 cells characterize nasal polyps with a default TH17 signature in nasal mucosa.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) in Western countries is characterized by eosinophilia, IgE production, and TH2 cytokine expression. Type 2 innate lymphoid cells from polyps produce IL-5 and IL-13 in response to IL-25 and IL-33, although the relevance of this axis to local mucosal T-cell responses is unknown. OBJECTIVE: We sought to investigate the role of the IL-25/IL-33 axis in local mucosal T-cell responses in patients with CRSwNP. METHODS: Polyp tissue and blood were obtained from patients undergoing nasal polypectomy. Control nasal biopsy specimens and blood were obtained from healthy volunteers. Tissue was cultured in a short-term explant model. T-cell surface phenotype/intracellular cytokines were assessed by means of flow cytometry. T-cell receptor variable ß-chain analysis was performed with the immunoSEQ assay. Microarrays were performed for gene expression analysis. RESULTS: IL-25 receptor (IL-17RB)-expressing TH2 effector cells were identified in nasal polyp tissue but not the healthy nasal mucosa or periphery. IL-17RB(+)CD4(+) polyp-derived TH2 cells coexpressed ST2 (IL-33 receptor) and responded to IL-25 and IL-33 with enhanced IL-5 and IL-13 production. Within IL-17RB(+)CD4(+) T cells, several identical T-cell receptor variable ß-chain complementarity-determining region 3 sequences were identified in different subjects, suggesting clonal expansion driven by a common antigen. Abundant IL-17-producing T cells were observed in both healthy nasal mucosal and polyp populations, with TH17-related genes the most overexpressed compared with peripheral blood T cells. CONCLUSION: IL-25 and IL-33 can interact locally with IL-17RB(+)ST2(+) polyp T cells to augment TH2 responses in patients with CRSwNP. A local TH17 response might be important in healthy nasal mucosal immune homeostasis.

Rhinovirus-induced IL-25 in asthma exacerbation drives type 2 immunity and allergic pulmonary inflammation.

Rhinoviruses (RVs), which are the most common cause of virally induced asthma exacerbations, account for much of the burden of asthma in terms of morbidity, mortality, and associated cost. Interleukin-25 (IL-25) activates type 2-driven inflammation and is therefore potentially important in virally induced asthma exacerbations. To investigate this, we examined whether RV-induced IL-25 could contribute to asthma exacerbations. RV-infected cultured asthmatic bronchial epithelial cells exhibited a heightened intrinsic capacity for IL-25 expression, which correlated with donor atopic status. In vivo human IL-25 expression was greater in asthmatics at baseline and during experimental RV infection. In addition, in mice, RV infection induced IL-25 expression and augmented allergen-induced IL-25. Blockade of the IL-25 receptor reduced many RV-induced exacerbation-specific responses including type 2 cytokine expression, mucus production, and recruitment of eosinophils, neutrophils, basophils, and T and non-T type 2 cells. Therefore, asthmatic epithelial cells have an increased intrinsic capacity for expression of a pro-type 2 cytokine in response to a viral infection, and IL-25 is a key mediator of RV-induced exacerbations of pulmonary inflammation.