Erwin W. Gelfand

Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator ( CFTR ) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes ( IL-1 β , CXCL8 , CREM ), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR -related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.

Background: Alpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), “augmentation therapy”, provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT. Methods: In an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo. Results: Both elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4⁺ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17. Conclusions: Compared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.

The impact of naturally occurring regulatory T cells (nTregs) on the suppression or induction of lung allergic responses in mice depends on the nuclear environment and the production of the pro-inflammatory cytokine interleukin 6 (IL-6). These activities were shown to be different in nTregs derived from wild-type (WT) and CD8-deficient mice (CD8^−/−), with increased IL-6 levels in nTregs from CD8^−/− mice in comparison to WT nTregs. Thus, identification of the molecular mechanisms regulating IL-6 production is critical to understanding the phenotypic plasticity of nTregs. Electrophoretic mobility shift assays (EMSA) were performed to determine transcription factor binding to four Il-6 promoter loci using nuclear extracts from nTregs of WT and CD8^−/− mice. Increased transcription factor binding for each of the Il-6 loci was identified in CD8^−/− compared to WT nTregs. The impact of transcription factor binding and a novel short tandem repeat (STR) on Il-6 promoter activity was analyzed by luciferase reporter assays. The Il-6 promoter regions closer to the transcription start site (TSS) were more relevant to the regulation of Il-6 depending on NF-κB, c-Fos, and SP and USF family members. Two Il-6 promoter loci were most critical for the inducibility by lipopolysaccharide (LPS) and tumor necrosis factor α (TNFα). A novel STR of variable length in the Il-6 promoter was identified with diverging prevalence in nTregs from WT or CD8^−/− mice. The predominant GT repeat in CD8^−/− nTregs revealed the highest luciferase activity. These novel regulatory mechanisms controlling the transcriptional regulation of the Il-6 promoter are proposed to contribute to nTregs plasticity and may be central to disease pathogenesis.

Activation of the steroidogenic enzyme CYP11A1 was shown to be necessary for the development of peanut-induced intestinal anaphylaxis and IL-13 production in allergic mice. We determined if levels of CYP11A1 in peripheral blood T cells from peanut-allergic (PA) children compared to non-allergic controls were increased and if levels correlated to IL-13 production and oral challenge outcomes to peanut. CYP11A1 mRNA and protein levels were significantly increased in activated CD4⁺ T cells from PA patients. In parallel, IL-13 production was significantly increased; IFNγ levels were not different between groups. There were significant correlations between expression levels of CYP11A1 mRNA and levels of IL13 mRNA and protein, levels of serum IgE anti-Ara h 2 and to outcomes of peanut challenge. The importance of CYP11A1 on cytokine production was tested using a CYP11A1 CRISPR/Cas9 KO plasmid or an inhibitor of enzymatic CYP11A1 activity. Inhibition of CYP11A1 activation in patient cells treated with the inhibitor, aminoglutethimide, or CD4⁺ T cell line transfected with the CYP11A1 KO plasmid resulted in reduced IL-13 production. These data suggest that the CYP11A1-CD4⁺Tcell-IL-13 axis in activated CD4⁺ T cells from PA children is associated with development of PA reactions. CYP11A1 may represent a novel target for therapeutic intervention in PA children.

The complexity of asthma is underscored by the number of cell types and mediators implicated in the pathogenesis of this heterogeneous syndrome. Type 2 CD4⁺T-cells (Th2) and more recently, type 2 innate lymphoid cells dominate current descriptions of asthma pathogenesis. However, another important source of these type 2 cytokines, especially interleukin (IL)-5 and IL-13, are CD8⁺T-cells, which are increasingly proposed to play an important role in asthma pathogenesis, because they are abundant and are comparatively insensitive to corticosteroids. Many common triggers of asthma exacerbations are mediatedviacorticosteroid-resistant pathways involving neutrophils and CD8⁺T-cells. Extensive murine data reveal the plasticity of CD8⁺T-cells and their capacity to enhance airway inflammation and airway dysfunction. In humans, Tc2 cells are predominant in fatal asthma, while in stable state, severe eosinophilic asthma is associated with greater numbers of Tc2 than Th2 cells in blood, bronchoalveolar lavage fluid and bronchial biopsies. Tc2 cells strongly express CRTH2, the receptor for prostaglandin D2, the cysteinyl leukotriene receptor 1 and the leukotriene B4 receptor. When activated, these elicit Tc2 cell chemotaxis and production of chemokines and type 2 and other cytokines, resulting directly or indirectly in eosinophil recruitment and survival. These factors position CD8⁺Tc2 cells as important and underappreciated effector cells contributing to asthma pathogenesis. Here, we review recent advances and new insights in understanding the pro-asthmatic functions of CD8⁺T-cells in eosinophilic asthma, especially corticosteroid-resistant asthma, and the molecular mechanisms underlying their pathologic effector function.