An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE(+)) cells is poorly understood. A model of the differentiation of IgE(+) B cells proposes that IgE(+) cells develop through a germinal-center IgG1(+) intermediate and that IgE memory resides in the compartment of IgG1(+) memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE(+) cells developed through a germinal-center IgE(+) intermediate to form IgE(+) memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.

The effects of inhaled corticosteroids on healthy airways.

BACKGROUND: The effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined. OBJECTIVES: To delineate the effects of ICS on gene expression in healthy airways, without confounding caused by changes in disease-related genes and disease-related alterations in ICS responsiveness. METHODS: Randomized open-label bronchoscopy study of high-dose ICS therapy in 30 healthy adult volunteers randomized 2:1 to (i) fluticasone propionate 500 mcg bd daily or (ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics. RESULTS: ICS induced small between-group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type-2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell-mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA-DQB2, CD96, PTPN7), B-cell immunity (CD20, immunoglobulin heavy and light chains) and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL-17-dependent gene signature was not upregulated by ICS. CONCLUSIONS: In healthy airways, 4-week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type-2 inflammation. This implies that homeostasis in health involves tonic type-2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.

Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Airway remodelling rather than cellular infiltration characterizes both type2 cytokine biomarker-high and -low severe asthma.

BACKGROUND: The most recognizable phenotype of severe asthma comprises people who are blood eosinophil and FeNO-high, driven by type 2 (T2) cytokine biology, which responds to targeted biological therapies. However, in many people with severe asthma, these T2 biomarkers are suppressed but poorly controlled asthma persists. The mechanisms driving asthma in the absence of T2 biology are poorly understood. OBJECTIVES: To explore airway pathology in T2 biomarker-high and -low severe asthma. METHODS: T2 biomarker-high severe asthma (T2-high, n = 17) was compared with biomarker-intermediate (T2-intermediate, n = 21) and biomarker-low (T2-low, n = 20) severe asthma and healthy controls (n = 28). Bronchoscopy samples were processed for immunohistochemistry, and sputum for cytokines, PGD2 and LTE4 measurements. RESULTS: Tissue eosinophil, neutrophil and mast cell counts were similar across severe asthma phenotypes and not increased when compared to healthy controls. In contrast, the remodelling features of airway smooth muscle mass and MUC5AC expression were increased in all asthma groups compared with health, but similar across asthma subgroups. Submucosal glands were increased in T2-intermediate and T2-low asthma. In spite of similar tissue cellular inflammation, sputum IL-4, IL-5 and CCL26 were increased in T2-high versus T2-low asthma, and several further T2-associated cytokines, PGD2 and LTE4 , were increased in T2-high and T2-intermediate asthma compared with healthy controls. CONCLUSIONS: Eosinophilic tissue inflammation within proximal airways is suppressed in T2 biomarker-high and T2-low severe asthma, but inflammatory and structural cell activation is present, with sputum T2-associated cytokines highest in T2 biomarker-high patients. Airway remodelling persists and may be important for residual disease expression beyond eosinophilic exacerbations. Registered at ClincialTrials.gov: NCT02883530.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

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.