The E3 ligase subunit FBXO45 binds the interferon-λ receptor and promotes its degradation during influenza virus infection.

Influenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ-IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses.

Inflammasome Activation in an In Vitro Sepsis Model Recapitulates Increased Monocyte Distribution Width Seen in Patients With Sepsis.

OBJECTIVES: Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis. DESIGN: An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer. SETTING: University research laboratory. SUBJECTS: Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D). INTERVENTIONS: In vitro treatment of specimens with bacterial LPS. MEASUREMENTS AND MAIN RESULTS: Wild-type THP1 cells demonstrated a significant increase in cell area (207 µm2 [159-400 µm2] vs 160 µm2 [134-198 µm2]; p < 0.001) and distribution width (198 vs 55 µm2; p < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1ß and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1ß release and increased MDW (median 24.7 U [22.2-27.2 U] vs 16.3 U [15.1-17.6 U]; p = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1ß release in the human subject samples as well. CONCLUSIONS: The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5's link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

In vitro 3D culture lung model from expanded primary cystic fibrosis human airway cells.

BACKGROUND: In vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators. METHODS: Primary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor). RESULTS: CF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661. CONCLUSIONS: CF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.

Reduced expression of the Ion channel CFTR contributes to airspace enlargement as a consequence of aging and in response to cigarette smoke in mice.

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease resulting in respiratory failure and represents the third leading cause of global death. The two classical phenotypes of COPD are chronic bronchitis and emphysema. Owing to similarities between chronic bronchitis and the autosomal-recessive disease Cystic Fibrosis (CF), a significant body of research addresses the hypothesis that dysfunctional CF Transmembrane Conductance Regulator (CFTR) is implicated in the pathogenesis of COPD. Much less attention has been given to emphysema in this context, despite similarities between the two diseases. These include early-onset cellular senescence, similar comorbidities, and the finding that CF patients develop emphysema as they age. To determine a potential role for CFTR dysfunction in the development of emphysema, Cftr+/+ (Wild-type; WT), Cftr+/- (heterozygous), and Cftr-/- (knock-out; KO) mice were aged or exposed to cigarette smoke and analyzed for airspace enlargement. Aged knockout mice demonstrated increased alveolar size compared to age-matched wild-type and heterozygous mice. Furthermore, both heterozygous and knockout mice developed enlarged alveoli compared to their wild-type counterparts following chronic smoke exposure. Taken into consideration with previous findings that cigarette smoke leads to reduced CFTR function, our findings suggest that decreased CFTR expression sensitizes the lung to the effects of cigarette smoke. These findings may caution normally asymptomatic CF carriers against exposure to cigarette smoke; as well as highlight emphysema as a future challenge for CF patients as they continue to live longer. More broadly, our data, along with clinical findings, may implicate CFTR dysfunction in a pathology resembling accelerated aging.

The psychoactive substance of cannabis Δ9-tetrahydrocannabinol (THC) negatively regulates CFTR in airway cells.

BACKGROUND: Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function. METHODS: Cultures of human bronchial epithelial cell line 16HBE14o- and primary human airway epithelial cells were exposed to THC. The expression of CFTR protein was determined by immunoblotting and CFTR function was measured using Ussing chambers. We also used specific pharmacological inhibitors of EGFR and ERK to determine the role of this pathway in THC-induced regulation of CFTR. RESULTS: THC decreased CFTR protein expression in primary human bronchial epithelial cells. This decrease was associated with reduced CFTR-mediated short-circuit currents. THC also induced activation of the ERK MAPK pathway via activation of EGFR. Inhibition of EGFR or MEK/ERK prevented THC-induced down regulation of CFTR protein expression. CONCLUSIONS AND GENERAL SIGNIFICANCE: THC negatively regulates CFTR and this is mediated through the EGFR/ERK axis. This study provides the first evidence that THC present in marijuana reduces the expression and function of CFTR in airway epithelial cells.