An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular Vesicles

Tumor-derived extracellular vesicles (TEV) “educate” healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy

Characterization of Inactivated Influenza Vaccines Used in the Russian National Immunization Program

Background: today’s standard quality control methods used to control the protein composition of inactivated influenza vaccines only take into account a few key reference components. They do not allow for thorough characterization of protein compositions. As a result, observation of unpredictable variations in major viral constituents and admixtures of cellular proteins within manufactured vaccines that may seriously influence the immunogenicity and safety of such vaccines has become a pressing issue in vaccinology. This study aims at testing a more sophisticated approach for analysis of inactivated split influenza vaccines licensed in the Russian Federation. The formulations under study are the most available on the market and are included in the Russian National Immunization Program. Methods: liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, in combination with label-free protein quantitation via the intensity-based absolute-quantitation (iBAQ) algorithm, as well as a number of standard molecular analysis methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM) were applied. Results: the methods implemented were able to identify dozens of viral and host proteins and quantify their relative amounts within the final formulations of different commercially available inactivated split influenza vaccines. Investigation of molecular morphology of the vaccine preparations using TEM revealed typical rosettes of major surface proteins (hemagglutinin and neuraminidase). DLS was used to demonstrate a size distribution of the rosettes and to test the stability of vaccine preparations at increased temperatures. Conclusions: a holistic approach based on modern, highly productive analytical procedures was for the first time applied for a series of different commercially available inactivated split influenza vaccines licensed in Russia. The protocols probed may be suggested for the post-marketing quality control of vaccines. Comparison of different preparations revealed that the Ultrix® and Ultrix® Quadri vaccines produced by pharmaceutical plant FORT LLC and trivalent vaccine Vaxigrip® produced by pharmaceutical company Sanofi Pasteur have well-organized antigen rosettes, they contain fewer admixture quantities of host cell proteins, and demonstrate good correlation among mostly abundant viral proteins detected by different methods

A BRISC-SHMT Complex Deubiquitinates IFNAR1 and Regulates Interferon Responses

Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here, we identify serine hydroxymethyltransferase (SHMT) as a previously unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub-mediated internalization and lysosomal degradation. BRISC-deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a mechanism of DUB regulation and suggest a therapeutic use of BRISC inhibitors for treating pathophysiological processes driven by elevated IFN responses

Suppression of Type I Interferon Signaling Overcomes Oncogene-Induced Senescence and Mediates Melanoma Development and Progression

Oncogene activation induces DNA damage responses and cell senescence. We report a key role of type I interferons (IFNs) in oncogene-induced senescence. IFN signaling-deficient melanocytes expressing activated Braf do not exhibit senescence and develop aggressive melanomas. Restoration of IFN signaling in IFN-deficient melanoma cells induces senescence and suppresses melanoma progression. Additional data from human melanoma patients and mouse transplanted tumor models suggest the importance of non-cell-autonomous IFN signaling. Inactivation of the IFN pathway is mediated by the IFN receptor IFNAR1 downregulation that invariably occurs during melanoma development. Mice harboring an IFNAR1 mutant, which is partially resistant to downregulation, delay melanoma development, suppress metastatic disease, and better respond to BRAF or PD-1 inhibitors. These results suggest that IFN signaling is an important tumor-suppressive pathway that inhibits melanoma development and progression and argue for targeting IFNAR1 downregulation to prevent metastatic disease and improve the efficacy of molecularly target and immune-targeted melanoma therapies

DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

Expression of type I interferons (IFNs) can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging

Triggering ubiquitination of IFNAR1 protects tissues from inflammatory injury

Abstract Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro. The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1SA knock‐in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1SA mice (or their bone marrow‐receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1SA mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation‐induced tissue damage can be purposely mimicked for therapeutic benefits