Lauren Mays

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Flagellin Induces Myeloid-Derived Suppressor Cells: Implications for Pseudomonas aeruginosa Infection in Cystic Fibrosis Lung Disease

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa–infected CF patient ex vivo–isolated as well as flagellin or P. aeruginosa in vitro–generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell–mediated host defense in CF and other P. aeruginosa–associated chronic lung diseases.

Neutrophilic myeloid-derived suppressor cells in cord blood modulate innate and adaptive immune responses

Neonates show an impaired anti‐microbial host defence, but the underlying immune mechanisms are not understood fully. Myeloid‐derived suppressor cells (MDSCs) represent an innate immune cell subset characterized by their capacity to suppress T cell immunity. In this study we demonstrate that a distinct MDSC subset with a neutrophilic/granulocytic phenotype (Gr‐MDSCs) is highly increased in cord blood compared to peripheral blood of children and adults. Functionally, cord blood isolated Gr‐MDSCs suppressed T cell proliferation efficiently as well as T helper type 1 (Th1), Th2 and Th17 cytokine secretion. Beyond T cells, cord blood Gr‐MDSCs controlled natural killer (NK) cell cytotoxicity in a cell contact‐dependent manner. These studies establish neutrophilic Gr‐MDSCs as a novel immunosuppressive cell subset that controls innate (NK) and adaptive (T cell) immune responses in neonates. Increased MDSC activity in cord blood might serve as key fetomaternal immunosuppressive mechanism impairing neonatal host defence. Gr‐MDSCs in cord blood might therefore represent a therapeutic target in neonatal infections.

Neutrophils express distinct RNA receptors in a non-canonical way

Background: RNAs modulate immune responses. Neutrophils represent the major fraction of immune cells, but receptors by which neutrophils sense RNA are poorly characterized. Results: Neutrophils and differentiated HL-60 cells express the RNA receptors RIG-I, MDA-5, and TLR8. RIG-I and MDA-5 are localized in secretory vesicles. Conclusion: Neutrophils express a distinct pattern of RNA receptors. Significance: RNA receptors on neutrophils could have implications for RNA-based therapeutics. RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n=442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤70% predicted, OR 7.24; p=2.30×10−5). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.

Expression and Regulation of Interferon-Related Development Regulator–1 in Cystic Fibrosis Neutrophils

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.