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(1) Background: Infection with human cytomegalovirus (HCMV) leads to the production and release of subviral particles, termed Dense Bodies (DB). They are enclosed by a membrane resembling the viral envelope. This membrane mediates the entrance of DBs into cells in a way that is comparable to virus infection. HCMV attachment and entry trigger the induction of interferon synthesis and secretion, and the subsequent expression of interferon-regulated genes (IRGs) that might inhibit replication of the virus. Recently, we demonstrated that DBs induce a robust interferon response in the absence of infection. Little is known thus far, including how DBs influence HCMV infection and virus–host interaction. (2) Methods: Purified DBs were used to study the impact on virus replication and on the innate defense mechanisms of the cell. (3) Results: The incubation of cells with DBs at the time of infection had little effect on viral genome replication. Preincubation of DBs, however, led to a marked reduction in viral release from infected cells. These cells showed an enhancement of the cytopathic effect, associated with a moderate increase in early apoptosis. Despite virus-induced mechanisms to limit the interferon response, the induction of interferon-regulated genes (IRGs) was upregulated by DB treatment. (4) Conclusions: DBs sensitize cells against viral infection, comparable to the effects of interferons. The activities of these particles need to be considered when studying viral–host interaction.

The flagellin (FliC) of Salmonella typhimurium is a potential vaccine adjuvant as it can activate innate immunity and promote acquired immune responses. Macrophages are an important component of the innate immune system. The mechanism of flagellin’s adjuvant activity has been shown to be related to its ability to activate macrophages. However, few studies have comprehensively investigated the effects of Salmonella flagellin in macrophages using transcriptome sequencing. In this study, RNA-Seq was used to analyze the expression patterns of RAW264.7 macrophages induced by FliC to identify novel transcriptomic signatures in macrophages. A total of 2204 differentially expressed genes were found in the FliC-treated group compared with the control. Gene ontology and KEGG pathway analyses identified the top significantly regulated functional classification and canonical pathways, which were mainly related to immune responses and regulation. Inflammatory cytokines (IL-6, IL-1β, TNF-α, etc.) and chemokines (CXCL2, CXCL10, CCL2, etc.) were highly expressed in RAW264.7 cells following stimulation. Notably, flagellin significantly increased the expression of interferon (IFN)-β. In addition, previously unidentified IFN regulatory factors (IRFs) and IFN-stimulated genes (ISGs) were also significantly upregulated. The results of RNA-Seq were verified, and furthermore, we demonstrated that flagellin increased the expression of IFN-β and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-β-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.

Depression may occur in patients with multiple sclerosis, especially during interferon-β (IFN-β) treatment, and therapy with antidepressants may be necessary. Interactions of IFN-β with antidepressants concerning glia-mediated inflammation have not yet been studied. Primary rat co-cultures of astrocytes containing 5% (M5, consistent with “physiological” conditions) or 30% (M30, consistent with “pathological, inflammatory” conditions) of microglia were incubated with 10 ng/mL amitriptyline or doxepin for 2 h, or with 2000 U/mL IFN-β for 22 h. To investigate the effects of antidepressants on IFN-β treatment, amitriptyline or doxepin was added to IFN-β pre-treated co-cultures. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to measure the glial cell viability, immunocytochemistry was performed to evaluate the microglial activation state, and ELISA was performed to measure pro-inflammatory TNF-α and IL-6 cytokine concentrations. Incubation of inflammatory astrocyte–microglia co-cultures with amitriptyline, doxepin or IFN-β alone, or co-incubation of IFN-β pre-treated co-cultures with both antidepressants, significantly reduced the extent of inflammation, with the inhibition of microglial activation. TNF-α and IL-6 levels were not affected. Accordingly, the two antidepressants did not interfere with the anti-inflammatory effect of IFN-β on astrocytes and microglia. Furthermore, no cytotoxic effects on glial cells were observed. This is the first in vitro study offering novel perspectives in IFN-β treatment and accompanying depression regarding glia.

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-β pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future., Graphical abstract, Dissecting the oncolytic property of uncharacterized viruses is pivotal in developing novel oncolytic vectors (OVs). Investigation into effects of Chandipura virus infection in cell lines of human origin and xenograft tumor model revealed marked susceptibility of U-138 glioma cells. Normal cells being relatively resistant offers the rationale for developing CHPV-OV.

AIM: To explore the expression of cGAS/STING signaling components in Mooren’s ulcer (MU). METHODS: Samples were obtained from ten MU patients, and eight residual corneal-scleral rings of healthy donor corneas for controls. Human corneal epithelial cells (HCECs) were used to evaluate the effect of cGAS/STING signaling pathway. Immunohistochemistry (IHC) and Western blot were used to examine the expression of cGAS, STING, and phosphorylated interferon regulatory factor 3 (p-IRF3) in MU tissues. The expression of interferon-β (IFN-β) and interferon-stimulated genes (ISGs) was quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The protein levels of cGAS and STING in MU samples were significantly elevated when compared with the healthy controls by Western blot and IHC. After stimulation with cGAMP, real-time PCR and ELISA showed a dramatic increase of IFN-β and ISGs (containing CXCL10, IFIT1, and IL-6) in HCECs. Moreover, HCECs treated with cGAMP was characterized by increased phosphorylation and more nuclear translocation of IRF3. Meanwhile, increased p-IRF3 was observed in MU samples via IHC and Western blot. CONCLUSION: The pronounced expression of cGAS/STING signaling components in the patients with MU and probably contribute to the onset and development of MU.

In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A viruses (IAVs) with low to high viral copy numbers in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating pro-inflammatory cytokines (TNF-α, IL-6, IFN-β) and antiviral interferon-stimulated genes (ISG-15, IFIM1, and TRIM22). A549 cells (3.0 × 105 cells) were inoculated with circulating seasonal IAV strains and incubated for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed using IAV-RNA copies in the culture supernatant and cell pellets to evaluate gene expression. At 24 h post-infection, cells were collected for IFN-β and ISG-15 protein expression. A549 cells inoculated with seasonal IAV strains with a high growth capability expressed lower levels of IFN-β and ISGs than strains with low growth capabilities. Moreover, suppression of the JAK/STAT pathway enhanced the viral copies of seasonal IAV strains with a low growth capability. Our results suggest that the expression of ISG-15, IFIM1, and TRIM22 in seasonal IAV-inoculated A549 cells could influence the regulation of viral replication, indicating the existence of strains with high and low growth capability. Our results may contribute to the development of new and effective therapeutic strategies to reduce the risk of severe influenza infections.

Background This investigator-initiated, open-label, single-arm, single-institute study was conducted to investigate the effectiveness of induction combination chemoradiotherapy and long-term maintenance therapy with temozolomide (TMZ) plus interferon (IFN)-β for glioblastoma. Methods The initial induction combination chemoradiotherapy comprised radiotherapy plus TMZ plus vincristine plus IFN-β. Maintenance chemotherapy comprised monthly TMZ, continued for 24–50 cycles, plus weekly IFN-β continued for as long as possible. The primary endpoint was 2-year overall survival (2y-OS). The study protocol was to be considered valid if the expected 2y-OS was over 38% and the lower limit of the 95% confidence interval (CI) was no less than 31.7% compared with historical controls, using Kaplan-Meier methods. Secondary endpoints were median progression-free survival (mPFS), median OS (mOS), 5-year OS rate (5y-OS), and mPFS and mOS classified according to MGMT promoter methylation status. Results Forty-seven patients were analyzed. The 2y-OS was 40.7% (95%CI, 27.5–55.4%). The mPFS and mOS were 11.0 months and 18.0 months, respectively, and 5y-OS was 20.3% (95%CI, 10.9–34.6%). The mPFS in groups with and without MGMT promoter methylation in the tumor was 10.0 months and 11.0 months (p = 0.59), respectively, and mOS was 24.0 months and 18.0 months (p = 0.88), respectively. The frequency of grade 3/4 neutropenia was 19.1%. Conclusions The 2y-OS with induction multidrug combination chemoradiotherapy and long-term maintenance therapy comprising TMZ plus IFN-β tended to exceed that of historical controls, but the lower limit of the 95%CI was below 31.7%. Although the number of cases was small, this protocol may rule out MGMT promoter methylation status as a prognostic factor. Trial registration University Hospital Medical Information Network (number UMIN000040599).

Severe acute respiratory syndrome coronavirus 2 principally weakens the hosts’ innate immune system by impairing the interferon function and production. Type I interferons (IFNs) especially IFN-β are best known for their antiviral activities. IFNs accompanied by the standard care protocols have opened up unique opportunities for treating the coronavirus disease 2019 (COVID-19). The databases including PubMed, SCOPUS, EMBASE, and Google Scholar were searched up to October 30, 2020. The primary and secondary outcomes were considered discharge and mortality, respectively. The abovementioned outcomes of standard care protocol were compared with the standard care plus IFN-β in the confirmed COVID-19 patients. Out of 356 records identified, 12 randomized clinical trial studies were selected for full-text screening. Finally, 5 papers were included in the systematic review and 3 papers in the meta-analysis. The average mortality rate was reported as 6.195% and 18.02% in intervention and control groups, respectively. Likewise, the median days of hospitalization were lower in the intervention group (9 days) than the control group (12.25 days). According to meta-analysis, IFN-β was found to increase the overall discharge rate (RR = 3.05; 95% CI: 1.09–5.01). Our findings revealed that early administration of IFN-β in combination with antiviral drugs is a promising therapeutic strategy against COVID-19.

Objective — to evaluate the compliance of interferon therapy in the presence of side effects in patients with multiple sclerosis.Methods and subjects. The analysis of following interferon therapy standard schemes in the presence of side effects was carried out at 48 patients with multiple sclerosis. The severity of the disease was determined using the Extended Disability Status Scale (EDSS). The average score on the EDSS scale was 4.30 ± 0.15 points. The drugs side effects were detected by the clinical and paraclinical examination and the registration cards filled in by the patients with a list of possible side reactions. Fatigue was scored by the Fatigue Severity Scale (FSS), and depression was assessed by The Beck Depression Inventory (BDI). Compliance was determined using the Moriski‑Green test.Results. Interferon therapy was carried out continuously in 22 (45.8 %) patients. There was a break in the treatment course in 18 (37.5 %), the drug was changed in 6 (12.5 %), and therapy was stopped in 2 (4.2 %) patients. Analysis of side effects as a potential factor in reducing adherence to treatment in non‑compliant patients revealed a predominance of post‑injection flu‑like syndrome and fatigue, which were observed in 96.2 % and 73.1 % of non‑compliant cases, respectively.Conclusions. The development of side effects of interferon in the form of influenza‑like syndrome and fatigue may affect compliance in patients with relapsing multiple sclerosis. Awareness of patients with potential side effects of INFb and measures for its prevention and treatment improve compliance with long‑term interferon therapy. Individualized approach and adequate management of interferoninduced side effects can improve the neuropsychological profile of patients with multiple sclerosis and increases the motivation to follow the recommended treatment regimen., Цель — оценить комплаенс интерферонотерапии при наличии побочного действия у больных рассеянным склерозом.Материалы и методы. Проведен анализ соблюдения стандартных схем интерферонотерапии при наличии побочного действия у 48 больных рассеянным склерозом. Степень тяжести проявлений заболевания устанавливали с помощью расширенной шкалы инвалидизации Курцтке (Expended Disability Status Scale, EDSS). Средний показатель по шкале EDSS составлял (4,30 ± 0,15) балла. Побочные эффекты препаратов выявляли по данным клинико‑параклинического обследования и заполненных пациентами регистрационных карт с перечнем возможных побочных реакций. Выраженность симптома усталости оценивали по шкале тяжести усталости (FSS), выраженность депрессии — с помощью шкалы депрессии Бека. Комплаенс определяли с помощью теста Мориски — Грина.Результаты. У 22 (45,8 %) больных интерферонотерапию проводили непрерывно, у 18 (37,5 %) — с перерывами разной продолжительности, у 6 (12,5 %) — со сменой препарата. Два 2 (4,2 %) пациента прекратили терапию. Анализ побочного действия как потенциального фактора уменьшения приверженности к лечению у некомплаентных пациентов выявил преобладание постинъекционного гриппоподобного синдрома и усталости, которые наблюдали в 96,2 и 73,1 % случаев соответственно.Выводы. Развитие побочных эффектов интерферона в виде гриппоподобного синдрома и усталости может влиять на терапевтический комплаенс у больных ремиттирующим рассеянным склерозом. Осведомленность пациентов о потенциальном побочном действии интерферона‑b и мерах по его профилактике и лечению улучшает комплаентность длительной интерферонотерапии. Индивидуальный подход и адекватный менеджмент в случае побочного действия препаратов интерферона могут улучшить нейропсихический профиль больных рассеянным склерозом и повысить мотивацию для соблюдения рекомендуемой схемы лечения., Мета — оцінити комплаєнс інтерферонотерапії за наявності побічної дії у хворих на розсіяний склероз.Матеріали і методи. Проведено аналіз дотримання стандартних схем інтерферонотерапії за наявності побічної дії у 48 хворих на розсіяний склероз. Ступінь тяжкості виявів захворювання встановлювали за допомогою розширеної шкали інвалідизації Курцтке (Expended Disability Status Scale, EDSS). Середній показник за шкалою EDSS становив (4,30 ± 0,15) бала. Побічні ефекти препаратів виявляли за даними клініко‑параклінічного обстеження і заповненої пацієнтом реєстраційної карти з переліком можливих побічних реакцій. Вираженість симптому втоми оцінювали за шкалою тяжкості втоми (FSS), вираженість депресії — за допомогою шкали депресії Бека. Комплаєнс визначали за допомогою тесту Моріскі — Гріна.Результати. У 22 (45,8 %) хворих інтерферонотерапію проводили безперервно, у 18 (37,5 %) — з перервами різної тривалості, у 6 (12,5 %) — зі зміною препарату. Два (4,2 %) пацієнти припинили терапію. Аналіз побічної дії як потенційного чинника зменшення прихильності до лікування у некомплаєнтних пацієнтів виявив переважання постін’єкційного грипоподібного синдрому і втоми, які спостерігали у 96,2 і 73,1 % випадків відповідно.Висновки. Розвиток побічної дії інтерферону у вигляді грипоподібного синдрому і втоми може впливати на терапевтичний комплаєнс у хворих на ремітивний розсіяний склероз. Обізнаність пацієнтів щодо потенційної побічної дії інтерферону‑b та заходів з її профілактики та лікування поліпшує комплаєнтність тривалої інтерферонотерапії. Індивідуальний підхід і адекватний менеджмент у разі побічної дії препаратів інтерферону можуть поліпшити нейропсихічний профіль хворих на розсіяний склероз та підвищити мотивацію для дотримання рекомендованої схеми лікування.

Background The mechanisms underlying the therapeutic activity of interferon-β in multiple sclerosis are still not completely understood. In the present study, we evaluated the short and long-term effects of interferon-β treatment on different subsets of regulatory T cells in relapsing–remitting multiple sclerosis patients biologically responsive to treatment because of mixovirus resistance protein A inducibility. Methods In this prospective longitudinal study, subsets of natural regulatory T cells (naïve, central memory and effector memory) and inducible regulatory T cells (Tr1), as well as in vitro-induced regulatory T cells (Tr1-like cells), were simultaneously quantified by flow cytometry in samples prepared from 148 therapy-naïve multiple sclerosis patients obtained before and after 6, 12, 18, and 24 months of interferon-β-1a treatment. mRNA for interleukin-10 and Tr1-related genes (CD18, CD49b, and CD46, together with Cyt-1 and Cyt-2 CD46-associated isoforms) were quantified in Tr1-like cells. Results Despite profound inter-individual variations in the modulation of all regulatory T-cell subsets, the percentage of natural regulatory T cells increased after 6, 12, and 24 months of interferon-β treatment. This increase was characterized by the expansion of central and effector memory regulatory T-cell subsets. The percentage of Tr1 significantly enhanced at 12 months of therapy and continued to be high at the subsequent evaluation points. Patients experiencing relapses displayed a higher percentage of naïve regulatory T cells and a lower percentage of central memory regulatory T cells and of Tr1 before starting interferon-β therapy. In addition, an increase over time of central memory and of Tr1 was observed only in patients with stable disease. However, in vitro-induced Tr1-like cells, prepared from patients treated for 24 months, produced less amount of interleukin-10 mRNA compared with pre-treatment Tr1-like cells. Conclusion Interferon-β induces the expansion of T regulatory subsets endowed with a high suppressive activity, especially in clinically stable patients. The overall concurrent modulation of natural and inducible regulatory T-cell subsets might explain the therapeutic effects of interferon-β in multiple sclerosis patients.

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasome-dependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in anti-flagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-β (IFN-β) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo. Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-β, but not for other pro-inflammatory cytokines. In addition, we found that anti-flagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-β produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

The anti-inflammatory activity of coffee extracts is widely recognized and supported by experimental evidence, in both in vitro and in vivo settings, mainly murine models. Here, we investigated the immunomodulatory properties of coffee extracts from green (GCE) and medium-roasted (RCE) Coffea canephora beans in human macrophages. The biological effect of GCE and RCE was characterized in LPS-stimulated THP-1-derived human macrophages (TDM) as a model of inflammation. Results showed decreased amounts of TNF-α, IL-6 and IL-1β and a strong dose-dependent inhibition of interferon-β (IFN-β) release. Molecular mechanism of IFN-β inhibition was further investigated by immunofluorescence confocal microscopy analysis that showed a diminished nuclear translocation of p-IRF-3, the main transcription factor responsible for IFN-β synthesis. The inhibition of IFN-β release by RCE and GCE was also confirmed in human primary CD14+ monocytes-derived macrophages (MDM). The main component of coffee extracts, 5-O-caffeoylquinic acid (5-CQA) also inhibited IFN-β production, through a mechanism occurring downstream to TLR4. Inhibition of IFN-β release by coffee extracts parallels with the activity of their main phytochemical component, 5-CQA, thus suggesting that this compound is the main responsible for the immunomodulatory effect observed. The application of 5-CQA and coffee derived-phytoextracts to target interferonopathies and inflammation-related diseases could open new pharmacological and nutritional perspectives.

A produção de glicoproteínas terapêuticas possui alto custo devido a necessidade de utilização de cultura de células de mamíferos que dependem de meios de cultivo complexos e caros e possuem baixo rendimento de produção. Leishmania tarentolae, um protozoário tripanossomátideo não patogênico para mamíferos, tem sido sugerido como um sistema alternativo para expressão heteróloga de glicoproteínas devido a existência de métodos eficientes de expressão heteróloga, ser facilmente adaptado para produção em alta escala e depender de meios de cultura de baixo custo. Além disso, este protozoário apresenta modificações pós- traducionais ausentes em bactérias e leveduras, organismos mais utilizados para produção industrial de proteínas recombinantes. O Interferon-β, é uma proteína utilizada em tratamentos de doenças como esclerose múltipla, as suas glicosilações são extremamente relevantes para suas funções biológicas. Embora o perfil de glicosilação de proteínas expressas em L. tarentolae seja semelhante a mamíferos, existem algumas diferenças no perfil de carboidratos presentes nas terminações das cadeias glicídicas devido à ausência de enzimas de biossínteses e incorporação de ácido siálico. Nesta tese foi desenvolvido e otimizado uma plataforma de expressão de glicoproteínas utilizando linhagens de L. tarentolae geneticamente otimizadas para síntese de glicoproteínas em alta escala mais estáveis com incorporação de ácido siálico na extremidade da cadeia glicídica e sua aplicação na produção de Interferon-β humano. Além da validação da produção do Interferon-β, também foi realizado caracterização de seus glicanos e validação de sua atividade para regulação de inflamação em modelo murinho, demonstrando que esta nova linhagem geneticamente otimizada de L. tarentolae é adequada para produção em larga escala de glicoproteínas terapêuticas. Palavras-chave: Leishmania tarentolae. Interferon-β. Proteínas terapêuticas. Glicosilação The production of therapeutic glycoproteins is expensive due to the need to use mammalian cell cultures that depend on complex and expensive culture media and have low yield of production. Leishmania tarentolae, a non-pathogenic mammalian trypanosomatid protozoan, has been suggested as an alternative system for heterologous expression of glycoproteins due to the existence of efficient methods of heterologous expression, it is easily adapted for high-scale production and depending on low-cost culture media. In addition, this protozoan presents post- translational modifications absent in bacteria and yeasts, organisms most used for industrial production of recombinant proteins. Interferon-β is a protein used in the treatment of diseases such as multiple sclerosis, its glycosylations are extremely relevant to its biological functions. Although the glycosylation profile of proteins expressed in L. tarentolae is similar to mammals, there are some differences in the profile of carbohydrates present at the ends of the sugar chains due to the absence of biosynthesis enzymes and sialic acid incorporation. Here, we developed and optimized a platform using genetically optimized L. tarentolae strains for the synthesis of more stable high-scale glycoproteins with sialic acid incorporation at the end of the sugar chain and its application in the production of human Interferon-β. In addition to validating the production of Interferon-β, characterization of its glycans and validation of its activity for regulating inflammation in a murine model was also performed, demonstrating that this new genetically optimized strain of L. tarentolae is suitable for large-scale production of glycoproteins therapeutics. Keywords: Leishmania tarentolae. Interferon-β. Therapeutic proteins. Glycosylation.

(1) Background: Cells infected with the human cytomegalovirus (HCMV) produce subviral particles, termed dense bodies (DBs), both in-vitro and in-vivo. They are released from cells, comparable to infectious virions, and are enclosed by a membrane that resembles the viral envelope and mediates the entry into cells. To date, little is known about how the DB uptake influences the gene expression in target cells. The purpose of this study was to investigate the impact of DBs on cells, in the absence of a viral infection. (2) Methods: Mass spectrometry, immunoblot analyses, siRNA knockdown, and a CRISPR-CAS9 knockout, were used to investigate the changes in cellular gene expression following a DB exposure; (3) Results: A number of interferon-regulated genes (IRGs) were upregulated after the fibroblasts and endothelial cells were exposed to DBs. This upregulation was dependent on the DB entry and mediated by the type I interferon signaling through the JAK-STAT pathway. The induction of IRGs was mediated by the sensing of the DB-introduced DNA by the pattern recognition receptor cGAS. (4) Conclusions: The induction of a strong type I IFN response by DBs is a unique feature of the HCMV infection. The release of DBs may serve as a danger signal and concomitantly contribute to the induction of a strong, antiviral immune response.

Background Interferon-β is an attractive drug for repurposing and use in the treatment of COVID-19, based on its in vitro antiviral activity and the encouraging results from clinical trials. The aim of this study was to analyze the impact of early interferon-β treatment in patients admitted with COVID-19 during the first wave of the pandemic. Methods This post hoc analysis of a COVID-19@Spain multicenter cohort included 3808 consecutive adult patients hospitalized with COVID-19 from 1 January to 17 March 2020. The primary endpoint was 30-day all-cause mortality, and the main exposure of interest was subcutaneous administration of interferon-β, defined as early if started ≤ 3 days from admission. Multivariate logistic and Cox regression analyses were conducted to identify the associations of different variables with receiving early interferon-β therapy and to assess its impact on 30-day mortality. A propensity score was calculated and used to both control for confounders and perform a matched cohort analysis. Results Overall, 683 patients (17.9%) received early interferon-β therapy. These patients were more severely ill. Adjusted HR for mortality with early interferon-β was 1.03 (95% CI, 0.82–1.30) in the overall cohort, 0.96 (0.82–1.13) in the PS-matched subcohort, and 0.89 (0.60–1.32) when interferon-β treatment was analyzed as a time-dependent variable. Conclusions In this multicenter cohort of admitted COVID-19 patients, receiving early interferon-β therapy after hospital admission did not show an association with lower mortality. Whether interferon-β might be useful in the earlier stages of the disease or specific subgroups of patients requires further research., This work was primarily supported by Fundación SEIMC/GeSIDA (grant number COVID-19/SEIMC-FSG). The funders had no role in study design, data collection, data analysis, data interpretation or writing of the manuscript. Additionally, IJ, JB, JRA, JRB, JC, and JP received funding for research from Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, cofinanced by the European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014–2020, through the following networks: Spanish AIDS Research Network (RIS) to IJ [grant number RD16CIII/0002/0006], JB [grant number RD16/0025/0017], and JRA [grant number RD16/0025/0018] and Spanish Network for Research in Infectious Diseases (REIPI) to JRB [grant number RD16/0016/0001], JC [grant number RD16/0016/0005], and JP [grant number RD16/0016/0009]. IJ [grant number CB21/13/00091], JB [grant number CB21/13/00044], JRA [grant number CB21/13/00039], JRB [grant number CB21/13/00012], and JC [grant number CB21/13/00009] also received support from the CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, cofinanced by the European Development Regional Fund.

Background and Aim Interferon‐stimulated gene 20 (ISG20) is an interferon‐inducible exonuclease that inhibits the replication of several RNA viruses. In patients with chronic hepatitis B, ISG20 expression is related to the interferon‐α treatment response. However, the molecular mechanism of ISG20‐mediated anti‐hepatitis B virus (HBV) activity is unclear. Methods We have investigated the effect of ISG20 on antiviral activity to address that. The life cycle of HBV was analyzed by the ectopic expression of ISG20 in HepG2 and HepG2‐NTCP cells. Finally, to provide physiological relevance of our study, the expression of ISG20 from chronic hepatitis B patients was examined. Results Interferon‐stimulated gene 20 was mainly induced by interferon‐β and dramatically inhibited HBV replication. In addition, ISG20 decreased HBV gene expression and transcription. Although ISG20 inhibited HBV replication by reducing viral enhancer activity, the expression of transcription factors that bind the HBV enhancer was not affected. Particularly, ISG20 suppressed HBV enhancer activity by binding to the enhancer II and core promoter (EnhII/Cp) region. Conclusion Our findings suggest that ISG20 exerts the anti‐HBV activity by acting as a putative repressor binding to the HBV EnhII/Cp region.

Type I IFN holds a critical role in host defence, providing protection against pathogenic organisms through coordinating a pro-inflammatory response. Type I IFN provides additional protection through mitigating this inflammatory response, preventing immunopathology. Within the context of viral infections, type I IFN signalling commonly results in successful viral clearance. Conversely, during bacterial infections, the role of type I IFN is less predictable, leading to either detrimental or beneficial outcomes. The factors responsible for the variability in the role of type I IFN remain unclear. Here, we aimed to elucidate differences in the effect of type I IFN signalling on macrophage functioning in the context of TLR activation. Using RAW 264.7 macrophages, we observed the influence of type I IFN to be dependent on the type of TLR ligand, length of TLR exposure and the timing of IFN-β signalling. However, in all conditions, IFN-β increased the production of the anti-inflammatory cytokine IL-10. Examination of RAW 264.7 macrophage function showed type I IFN to induce an activated phenotype by up-regulating MHC II expression and enhancing killing activity. Our results support a context-dependent role for type I IFN in regulating RAW 264.7 macrophage activity.

Multipl Skleroz (MS) hastalığının seyrinde ve tedavi sürecinde homosistein, vitamin B12 ve folik asit düzeylerindeki değişiklikler bildirilmiş ancak tam mekanizması aydınlatılamamıştır. Çalışmamızda, akut atak ile başvuran hastalarda yüksek doz metilprednizolon tedavisi öncesinde, sonrasında ve interferon-β tedavisi sonrasında serum B12, folik asit ve homosisitein düzeylerindeki değişiklikler incelenmiştir. Çalışmaya 1 yıl içinde akut atak ile başvuran, klinik kesin MS tanısı konarak takip edilen 31 hasta (8 erkek, 23 kadın) ve 10 kontrol olgusu dahil edildi. 20 hasta akut atak ile başvurdu 7 gün süreyle 1000 mg/gün metilprednizolon uygulandı. 27 hastaya interferon-β tedavisi başlandı. Yüksek doz steroid tedavi sonrası 1. ay düzeyleri ve interferon-β tedavisi alan hastaların 3. ay sonundaki serum vitamin B12, folik asit ve plazma homosistein düzeyleri ölçülerek kontrol grubu ve tedavisi öncesi düzeyleri ile karşılaştırıldı. Kontrol grubuna göre MS hastalarının bazal B12 değerleri daha önceki bulgularla tutarlı olarak daha düşüktü. Hem metilprednizolon, hem de interferon-β tedavisi sonrası B12 ve folik asid değerlerinde yükselme saptandı ancak sadece metilprednizolon tedavisi sonrasındaki artış istatiksel olarak anlamlıydı. Çalışmamızda saptanan metilprednizolon sonrası yükselen B12 ve folik asid düzeyleri ve istatistiksel anlamlılığa ulaşmayan homosistein düzeylerindeki düşme eğilimi farklı tartışmalar yaratmaktadır.

Background Interferon-β (IFN-β) is a cytokine with pleiotropic cellular functions, including antiviral, antiproliferative, and immunomodulatory activities. IFN-β inhibits multiple tumor cell growth in vitro. However, the contradiction between the therapeutic dose of IFN-β and its maximally tolerated dose is still inextricable in vivo. Human gingiva-derived mesenchymal stromal cells (GMSCs) represent promising vehicles for cancer gene therapy. This study evaluated the potential of GMSCs genetically engineered to produce IFN-β as a targeted gene delivery system to treat tongue squamous cell carcinoma (TSCC) in vitro and in vivo. Methods A lentiviral vector encoding IFN-β was constructed and transfected into GMSCs to obtain IFN-β gene-modified GMSCs (GMSCs/IFN-β). Enzyme-linked immunosorbent assay (ELISA) was used to measure the IFN-β concentration in conditioned medium (CM) from GMSCs/IFN-β. The Cell Counting Kit-8 (CCK8), colony formation assay, and flow cytometry were used to detect the effects of GMSCs/IFN-β on TSCC cell line CAL27 cell growth and apoptosis in vitro. TSCC xenograft model was developed by subcutaneous injection of CAL27 cells into BALB/c nude mouse, and the role of intravenously injected GMSCs/IFN-β in engrafting in TSCC and controlling tumor progression was measured in vivo. Results GMSCs/IFN-β expressed a high level of IFN-β. Both CCK8 and colony forming assay showed that GMSCs/IFN-β significantly inhibited the proliferation of CAL27 cells compared with the GMSCs, GMSCs/vector, or DMEM group. Flow cytometry analysis demonstrated that the CAL27 cell apoptosis rate was higher in the GMSCs/IFN-β group than in the other three groups. The in vivo experiment revealed that GMSCs/IFN-β engrafted selectively in TSCC xenograft and expressed a high level of IFN-β. There were smaller tumor volume and lower number of Ki67-positive cells in the GMSCs/IFN-β group than in the GMSCs, GMSCs/vector, or phosphate-buffered saline (PBS) group. Interestingly, GMSCs and GMSCs/vector also presented the potential of CAL27 cell growth inhibition in vitro and in vivo, although such an effect was weaker than GMSCs/IFN-β. Conclusions GMSCs/IFN-β inhibits the proliferation of TSCC cells in vitro and in vivo. These results provide evidence that delivery of IFN-β by GMSCs may be a promising approach to develop an effective treatment option for TSCC therapy.

Background Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity. Its expression level is increased in activated immune cells including dendritic cells, macrophages, and microglia. We have previously reported that CatH deficiency impairs toll-like receptor 3 (TLR3)-mediated activation of interferon regulatory factor 3 (IRF3), and the subsequent secretion of interferon (IFN)-β from dendritic cells. Furthermore, there is increasing evidence that IFN-β secreted from microglia/macrophages has neuroprotective effects. These observations prompted further investigation into the effects of CatH deficiency on neuropathological changes. Methods In this study, neuropathological changes were examined using histochemical staining (both hematoxylin-eosin (H&E) and Nissl) of the hippocampus of wild-type (WT) and CatH-deficient (CatH−/−) mice after hypoxia-ischemia (HI). The density and the localization of CatH and TLR3 were examined by immunofluorescent staining. CatH processing in microglia was assayed by pulse-chase experiments, while immunoblotting was used to examine TLR3 expression and IRF3 activation in microglia/macrophages in the presence of poly(I:C). Microglial cell death was examined by fluorescence-activated cell sorting (FACS), and primary astrocyte proliferation in the presence of IFN-β was examined using scratch wound assay. Results WT mice displayed severe atrophy in association with neuronal death and moderate astrogliosis in the hippocampus following neonatal HI. Somewhat surprisingly, CatH−/− mice showed marked neuronal death without severe atrophy in the hippocampus following HI. Furthermore, there was notable microglia/macrophages cell death and strong astrogliosis in the hippocampus. The TLR3 and phosphorylated IRF3 expression level in the hippocampus or splenocytes (mainly splenic macrophages); from CatH−/− mice was lower than in WT mice. In vitro experiments demonstrated that recombinant IFN-β suppressed HI-induced microglial cell death and astrocyte proliferation. Conclusion These observations suggest that CatH plays a critical role in the proteolytic maturation and stabilization of TLR3, which is necessary for IFN-β production. Therefore, impaired TLR3/IFN-β signaling resulting from CatH deficiency may induce microglial cell death after activation and astrogliosis/glial scar formation in the hippocampus following HI injury, leading to suppression of hippocampal atrophy.

In-transit metastases (ITMs) in patients with malignant melanoma (MM) are associated with poor prognosis and a worse disease burden compared with MM without ITMs. A substantial population of patients with ITMs show no or only poor responses to newly developed therapies, such as immune checkpoint inhibitors or molecular-targeted agents. It is difficult to control the exudate and bleeding from ITMs when these medications are ineffective. In Japan, local injection of interferon-β (IFN-β) has been licensed for years as adjuvant therapy for MM. However, the evidence for IFN-β effectiveness for ITMs remains low. The present report describes a case of MM with multiple ITMs that did not respond to a programmed cell death-1 inhibitor and local injections of IFN-β at 3 million IU/day for 5 days/4 weeks but remitted upon increasing the amount of IFN-β injections to 10 consecutive days/4 weeks. Local IFN-β therapy could be an option for improving the quality of life of patients.

Human norovirus (HuNoV) is the leading cause of epidemic acute gastroenteritis worldwide. Type I interferons (IFN)-α/β are highly potent cytokines that are initially identified for their essential roles in antiviral defense. It was reported that HuNoV infection did not induce IFN-β expression but was controlled in the presence of IFN-β in human intestinal enteroids and a gnotobiotic pig model, suggesting that HuNoV has likely developed evasion countermeasures. In this study, we found that a cDNA clone of GII.4 HuNoV, the predominantly circulating genotype worldwide, inhibits the production of IFN-β and identified the viral NTPase as a key component responsible for such inhibition. HuNoV NTPase not only inhibits the activity of IFN-β promoter but also the mRNA and protein production of IFN-β. Additional studies indicate that NTPase inhibits the phosphorylation and nuclear translocation of interferon-regulatory factor-3 (IRF-3), leading to the suppression of IFN-β promoter activation. Mechanistically, NTPase interacts with IkB kinase ε (IKKε), an important factor for IRF-3 phosphorylation, and such interaction blocks the association of IKKε with unanchored K48-linked polyubiquitin chains, resulting in the inhibition of IKKε phosphorylation. Further studies demonstrated that the 1-179 aa domain of NTPase which interacts with IKKε is critical for the suppression of IFN-β production. Our findings highlight the role of HuNoV NTPase in the inhibition of IFN-β production, providing insights into a novel mechanism underlying how HuNoV evades the host innate immunity.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-β (IFN-β) activation in IFN-β promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-β promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-β production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-β promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-β antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-β activation. However, most of these results were generated from IFN-β promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-β promoter luciferase activity, it showed no inhibitory effect on IFN-β production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-β signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

Although fingolimod and interferon-β are two mechanistically different multiple sclerosis (MS) treatments, they both induce B cell activating factor (BAFF) and shift the B cell pool towards a regulatory phenotype. However, whether there is a shared mechanism between both treatments in how they influence the B cell compartment remains elusive. In this study, we collected a cross-sectional study population of 112 MS patients (41 untreated, 42 interferon-β, 29 fingolimod) and determined B cell subsets, cell-surface and RNA expression of BAFF-receptor (BAFF-R) and transmembrane activator and cyclophilin ligand interactor (TACI) as well as plasma and/or RNA levels of BAFF, BAFF splice forms and interleukin-10 (IL-10) and -35 (IL-35). We added an in vitro B cell culture with four stimulus conditions (Medium, CpG, BAFF and CpG+BAFF) for untreated and interferon-β treated patients including measurement of intracellular IL-10 levels. Our flow experiments showed that interferon-β and fingolimod induced BAFF protein and mRNA expression (P ≤ 3.15 x 10-4) without disproportional change in the antagonizing splice form. Protein BAFF correlated with an increase in transitional B cells (P = 5.70 x 10-6), decrease in switched B cells (P = 3.29 x 10-4), and reduction in B cell-surface BAFF-R expression (P = 2.70 x 10-10), both on TACI-positive and -negative cells. TACI and BAFF-R RNA levels remained unaltered. RNA, plasma and in vitro experiments demonstrated that BAFF was not associated with increased IL-10 and IL-35 levels. In conclusion, treatment-induced BAFF correlates with a shift towards transitional B cells which are enriched for cells with an immunoregulatory function. However, BAFF does not directly influence the expression of the immunoregulatory cytokines IL-10 and IL-35. Furthermore, the post-translational mechanism of BAFF-induced BAFF-R cell surface loss was TACI-independent. These observations put the failure of pharmaceutical anti-BAFF strategies in perspective and provide insights for targeted B cell therapies.

Introduction: COVID-19 has several overlapping phases. Treatment has focused on the late stage of the disease in hospital. Yet, the continuation of the pandemic is by propagation of the disease in outpatients. The current public health strategy relies solely on vaccines to prevent disease. Areas Covered: We searched the major national registries, pubmed.org, and the preprint servers for all ongoing, completed and published trial results with subject numbers of 100 or more on, and used a targeted search to find announcements of unpublished trial results. As of 2/15/2021, we found 111 publications reporting findings in human studies on 14 classes of agents, and on 9 vaccines. There were 62 randomized controlled studies, the rest retrospective observational analyses. Only 21 publications dealt with outpatient care, the rest all in hospitalized patients. Remdesivir and convalescent plasma have emergency use authorization for hospitalized patients in the U.S.A. There is also support for glucocorticoid treatment of the COVID-19 respiratory distress syndrome. Monoclonal antibodies are authorized for outpatients, but the supply is inadequate to treat all at time of diagnosis. Favipiravir, ivermectin, and interferons are approved in certain countries Expert Opinion: Worldwide vaccination is now underway. Vaccines and antibodies are highly antigen specific and new variants are appearing. There is a need for treatment of outpatients who contract the disease, in addition to mass immunization. We call on public health authorities to authorize treatments with known low risk and potential benefit for use in parallel with mass immunization.

Objective The aim of this systematic review and meta-analysis of randomized controlled trials (RCTs) was to investigate the efficacy of interferon (IFN)-β-containing regimens in treating patients with COVID-19. Methods PubMed, Embase, Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov were searched from inception to July 17, 2021. RCTs comparing the clinical efficacy and safety of IFN-β-containing regimens (study group) to other antiviral treatment options or placebo (control group) in treating patients with COVID-19 were included. Results : Eight RCTs were included. No significant difference in the 28-day all -cause mortality rate was observed between the study and control groups (OR, 0.74; 95% CI, 0.44-1.24; I2 = 51%). The study groups had a lower rate of intensive care unit (ICU) admissions than the control groups (OR 0.58, 95% CI 0.36-0.95; I2 = 0%). Furthermore, INF-β was not associated with an increased risk of any adverse event (AE) or serious AE when compared with the control group. Conclusions IFN-β does not appear to provide an increased survival benefit in hospitalized patients with COVID-19, but may help reduce the risk of ICU admission. Moreover, IFN-β is a safe agent for use in the treatment of COVID-19.

BackgroundNeutralizing anti-drug antibodies (NAbs) to interferon beta (IFNβ) develop in up to 47% of multiple sclerosis (MS) treated patients inhibiting treatment effect of IFNβ. However, the long-term effect of NAbs remain unknown.ObjectiveTo investigate the long-term consequences of high titer NAbs to IFNβ on disease activity and progression in MS patients.MethodsAn observational study including data from all IFNβ treated relapsing remitting MS patients with sufficient NAb test results from the Swedish MS registry. Patients were classified into either confirmed ‘high titer’ or ‘persistent negative’ groups and analyzed for differences in disease activity and progression over time.ResultsA total of 197 high-titer and 2907 persistent negative patients with 19969.6 follow up years of data were included. High titer NAbs were associated with a higher degree of disease activity at baseline. However, even when accounting for this, the presence of high titer NAbs were also associated with higher disease activity during IFNβ treatment. This persisted even after the next DMT start, suggesting that earlier high titers may partially reduce the effect of later treatments. No difference was found in confirmed disability progression.ConclusionHigh titer NAbs to IFNβ are associated with higher disease activity, persisting even after IFNβ discontinuation or switch. These results support use of highly efficient treatment earlier in patients with active disease, to avoid these complications.

Background Neutralizing anti-drug antibodies (NAbs) to interferon beta (IFNβ) develop in up to 47% of multiple sclerosis (MS) treated patients inhibiting treatment effect of IFNβ. However, the long-term effect of NAbs remain unknown. Objective To investigate the long-term consequences of high titer NAbs to IFNβ on disease activity and progression in MS patients. Methods An observational study including data from all IFNβ treated relapsing remitting MS patients with sufficient NAb test results from the Swedish MS registry. Patients were classified into either confirmed ‘high titer’ or ‘persistent negative’ groups and analyzed for differences in disease activity and progression over time. Results A total of 197 high-titer and 2907 persistent negative patients with 19969.6 follow up years of data were included. High titer NAbs were associated with a higher degree of disease activity at baseline. However, even when accounting for this, the presence of high titer NAbs were also associated with higher disease activity during IFNβ treatment. This persisted even after the next DMT start, suggesting that earlier high titers may partially reduce the effect of later treatments. No difference was found in confirmed disability progression. Conclusion High titer NAbs to IFNβ are associated with higher disease activity, persisting even after IFNβ discontinuation or switch. These results support use of highly efficient treatment earlier in patients with active disease, to avoid these complications.

In December 2019, an increasing number of cases of novel coronavirus (2019-nCoV) that were linked to seafood wholesale market have identified in Wuhan, China. Taxonomist declared 2019-nCoV as severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) and the disease termed as COVID-19. Importantly, there is no approved drugs or vaccine against SARS-CoV-2. Current review is related to address treatment option for COVID-19. Interferon-β have shown significant activity against previous outbreak of SARS-CoV and MERS-CoV. Faced with an ongoing pandemic of SARS-CoV-2 and lack of effective treatment options, identifying compounds with antiviral activity has become a high priority. Efficacy of these treatments is still unclear. Therefore, final analysis is important to develop better treatment strategies for ongoing pandemic.

B cells are considered major contributors to multiple sclerosis (MS) pathophysiology. While lately approved disease-modifying drugs like ocrelizumab deplete B cells directly, most MS medications were not primarily designed to target B cells. Here, we review the current understanding how approved MS medications affect peripheral B lymphocytes in humans. These highly contrasting effects are of substantial importance when considering these drugs as therapy for neuromyelitis optica spectrum disorders (NMOSD), a frequent differential diagnosis to MS, which is considered being a primarily B cell- and antibody-driven diseases. Data indicates that MS medications, which deplete B cells or induce an anti-inflammatory phenotype of the remaining ones, were effective and safe in aquaporin-4 antibody positive NMOSD. In contrast, drugs such as natalizumab and interferon-β, which lead to activation and accumulation of B cells in the peripheral blood, lack efficacy or even induce catastrophic disease activity in NMOSD. Hence, we conclude that the differential effect of MS drugs on B cells is one potential parameter determining the therapeutic efficacy or failure in antibody-dependent diseases like seropositive NMOSD.

During the resolution of acute inflammation, macrophages undergo reprogramming from pro-inflammatory, to anti-inflammatory/reparative, and eventually to pro-resolving macrophages. Galectin-1 (Gal-1) is a bona fide pro-resolving lectin while interferon β (IFN-β) was recently shown to facilitate macrophage reprogramming and resolution of inflammation. In this study, we found Gal-1null mice exhibit a hyperinflammatory phenotype during the resolution of zymosan A-induced peritonitis but not during the early inflammatory response. This phenotype was characterized by reduced macrophage numbers, increased secretion of pro-inflammatory cytokines, such as interleukin-12 (IL-12), and reduced secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10). In addition, we found a delayed expression of the pro-resolving enzyme 12/15-lipoxygenase in macrophages and heightened levels of the inflammatory protease proteinase-3 (PR3) in peritoneal fluids from Gal-1null mice. Moreover, we observed sex-dependent differences in the inflammatory profile of Gal-1null mice. Notably, we found that IFN-β levels were reduced in resolution-phase exudates from Gal-1null mice. Administration of IFN-β in vivo or ex vivo treatment was able to rescue, at least in part, the hyperinflammatory profile of Gal-1null mice. In particular, IFN-β recovered a subset of F4/80+GR-1+ macrophages, restored IL-12 and IL-10 secretion from macrophages to WT values and diminished abnormal peritoneal PR3 levels in Gal-1null mice. In conclusion, our results revealed a new Gal-1-IFN-β axis that facilitates the resolution of inflammation and might restrain uncontrolled inflammatory disorders. Fil: Yaseen, Hiba. University of Haifa; Israel Fil: Butenko, Sergei. University of Haifa; Israel Fil: Polishuk Zotkin, Irina. University of Haifa; Israel Fil: Schif Zuck, Sagie. University of Haifa; Israel Fil: Pérez Sáez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ariel, Amiram. University of Haifa; Israel

Background: Multiple Sclerosis (MS) is one of the most common causes of neurological disability in young and middle-aged adults. Patients with MS face many challenges, both physical and emotional, and see an overall reduction in their autonomy. There is no definitive treatment for MS, though Disease Modifying Drugs (DMDs) have proved effective in reducing the frequency and severity of relapses. Unfortunately, long-term adherence to these therapies is a significant challenge due to practical difficulties as well as a general distrust towards the drugs . Objective: This study follows an original research carried out in 2008. In the first study, patients answered questions on their clinical history and expressed their judgment on the pharmacological treatment, their perceived effectiveness and factors that may undermine compliance. They have been recalled after two years to verify if the reported symptoms have changed and to assess how their knowledge of the disease and “acceptance” of the treatment have been modified. Methods: In spite of the relatively high number of patients participating in the first study (141 patients followed at a single neurological centre), only 16 patients have completed the questionnaire for the long-term survey. A detailed descriptive analysis has been carried out, as well as a pairwise correlation analysis. Results and Conclusions: The interviews carried out gave an insight into how patients’ behavior may have changed over time. Compliance rate is different in newly-diagnosed patients and long-term patients; the latter are more likely to be compliant, given their personal experience with the disease. Communications with neurologists and health personnel should aim at forming therapeutic alliances with patients and detecting their preferences for a qualitatively adequate assistance throughout their illness.

Coxsackievirus B3 (CVB3) is the primary etiologic agent of viral myocarditis, a major heart disease that occurs predominantly in children and young adolescents. In the heart, intercalated disks (ICD) are important structural formations that connect adjacent cardiomyocytes to maintain cardiac architecture and mediate signal communication. Deficiency in ICD components, such as desmosome proteins, leads to heart dysfunction. γ-catenin, a component protein of desmosomes, normally binds directly to desmocollin-2 and desmoglein-2. In this study, we found that CVB3 infection downregulated γ-catenin at the protein level but not the mRNA level in mouse HL-1 cardiomyocytes. We further found that this reduction of γ-catenin protein is a result of ubiquitin proteasome-mediated degradation, since the addition of proteasome inhibitor MG132 inhibited γ-catenin downregulation. In addition, we found that desmocollin-2 and desmoglein-2 were cleaved by both viral protease 3C and virus-activated cellular caspase, respectively. These cleavages led to the release of bound γ-catenin from the desmosome into the cytosol, resulting in rapid degradation of γ-catenin. Since γ-catenin shares high sequence homology with β-catenin in binding the TCF/LEF transcription factor, we further studied the effect of γ-catenin degradation on Wnt/β-catenin signaling. Luciferase assay showed that γ-catenin expression inhibited Wnt/β-catenin signaling. This finding was substantiated by qPCR to show that overexpression of γ-catenin downregulated transcription of Wnt signal target genes, c-myc and MMP9, while silencing γ-catenin upregulated these target genes. Finally, we demonstrated that γ-catenin expression inhibited CVB3 replication. In search for the underlying mechanism, we found that silencing γ-catenin caused down-regulation of interferon-β and its stimulated antiviral genes MDA5, MAVS, and ISG15. Taken together, our results indicate, for the first time, that CVB3 infection causes cardiomyocyte death through, at least in part, direct damage to the desmosome structure and reduction of γ-catenin protein, which in return promotes Wnt/β-catenin signaling and downregulates interferon-β stimulated immune responses.

Background: High serum levels of 25-hydroxyvitamin D (25(OH)D) have been found among patients with a favorable disease course in relapsing-remitting MS (RRMS), indicating that this may limit clinical deterioration. Clinical deterioration in RRMS correlates with increasing serum levels of neurofilament light chain (NfL). Objectives: To examine the association between physiological variations in serum 25(OH)D and NfL levels in RRMS patients before and during disease modifying therapy (DMT). Material and Methods: Serum 25(OH)D and NfL concentrations were measured in 85 newly diagnosed RRMS patients enrolled in a 24-month randomized double-blinded placebo-controlled trial of ω-3 fatty acid supplementation without vitamin D. Patients were without DMT until interferon β-1a (IFN-β) initiation at study month 6. Longitudinal serum measurements and brain magnetic resonance imaging (MRI) were obtained. Associations between 25(OH)D and NfL levels were analyzed with linear regression models for the whole study period and the periods before and during IFN-β treatment. Analyses with adjustment for inflammatory MRI disease activity were also performed. Results: No significant associations were found between variations in 25(OH)D and NfL levels during the whole study period (p = 0.95), or the periods without (p = 0.78) or with (p = 0.33) IFN-β therapy. Patients with inflammatory MRI disease activity had significantly higher serum NfL levels than patients without inflammatory MRI disease activity [mean (SD) difference 12.6 (2.0) pg/mL, p < 0.01]. Adjustment for this did not change the relationship between 25(OH)D and NfL concentrations. Conclusion: Natural variations in serum 25(OH)D values do not seem to be associated with alterations in serum NfL concentrations in RRMS patients. publishedVersion

Chronic activation of microglia is associated with retinal degeneration, which makes them a potential therapeutic target for retinal degenerative diseases including age-related macular degeneration (AMD). Interferon-beta (IFN-β) is a potent immune regulator, commonly used for the treatment of multiple sclerosis patients. We have previously shown that IFN-β prevents microgliosis and choroidal neovascularization in a laser model of wet AMD. Here, we hypothesized that microglia modulation via IFN-β may also dampen mononuclear phagocyte reactivity and thereby protect from retinal degeneration in a light-damage paradigm mimicking some features of dry AMD. BALB/cJ mice received intraperitoneal injections of 10,000 U IFN-β or vehicle every other day; starting at the day of exposure to 15,000 lux white light for 1 h. Systemic treatment with IFN-β partially enhanced IFN-α/β receptor (IFNAR) signaling in the retina and reduced the number of reactivated microglia in the subretinal space. However, four days after light damage neither decreased expression of complement factors nor rescue of retinal thickness was found. We conclude that IFNAR signaling modulate retinal microglia but cannot prevent strong retinal degeneration as elicited by acute white light damage., Highlights • Intraperitoneal application of IFN-β increases IFNAR signaling in the retina. • IFN-β administration reduces microglia migration to the subretinal space. • Light-induced retinal degeneration is not restored by IFN-β therapy.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory infections in infants and young children, accounting for an estimated 3 million hospitalizations annually worldwide. Despite the major health burden, there is currently no licensed RSV vaccine. RSV is recognized by a range of cellular receptors including both toll-like receptors (TLR) and retinoic acid-inducible gene-I-like receptors (RIG-I). This interaction initiates signaling through mitochondrial antiviral signaling (MAVS) and interferon regulatory factor (IRF) proteins, resulting in the induction of type I interferons (IFN). Early viral control is mediated by either IFN-α or IFN-β signaling through the IFN receptor (IFNAR), inducing the production of antiviral interferon-stimulating genes (ISGs). Type I IFNs also initiate the early production of proinflammatory cytokines including interleukin 6 (IL-6), tumor necrosis factor (TNF), and IFN-γ. Type I IFN levels correlate with age, and inadequate production may be a critical factor in facilitating the increased RSV disease severity observed in infants. Here, we review the current literature on the function of type I IFNs in RSV pathogenesis, as well as their involvement in the differential immune responses observed in infants and adults.

Cyclic di-AMP (c-di-AMP) is an important signaling molecule for pneumococci, and as a uniquely prokaryotic product it can be recognized by mammalian cells as a danger signal that triggers innate immunity. Roles of c-di-AMP in directing host responses during pneumococcal infection are only beginning to be defined. We hypothesized that pneumococci with defective c-di-AMP catabolism due to phosphodiesterase deletions could illuminate roles of c-di-AMP in mediating host responses to pneumococcal infection. Pneumococci deficient in phosphodiesterase 2 (Pde2) stimulated a rapid induction of interferon β (IFNβ) expression that was exaggerated in comparison to that induced by wild type (WT) bacteria or bacteria deficient in phosphodiesterase 1. This IFNβ burst was elicited in mouse and human macrophage-like cell lines as well as in primary alveolar macrophages collected from mice with pneumococcal pneumonia. Macrophage hyperactivation by Pde2-deficient pneumococci led to rapid cell death. STING and cGAS were essential for the excessive IFNβ induction, which also required phagocytosis of bacteria and triggered the phosphorylation of IRF3 and IRF7 transcription factors. The select effects of Pde2 deletion were products of a unique role of this enzyme in c-di-AMP catabolism when pneumococci were grown on solid substrate conditions designed to enhance virulence. Because pneumococci with elevated c-di-AMP drive aberrant innate immune responses from macrophages involving hyperactivation of STING, excessive IFNβ expression, and rapid cytotoxicity, we surmise that c-di-AMP is pivotal for directing innate immunity and host-pathogen interactions during pneumococcal pneumonia.

Although first-line chemotherapy has a high rate of complete responses in ovarian cancer patients, the vast majority of patients present with recurrent disease that has become refractory to conventional chemotherapy. Peritoneal dissemination and malignant ascites are the hallmarks of recurrent or advanced ovarian cancer and severely reduce quality of life. Development of therapeutic measures to treat such patients is eagerly anticipated. Macrophage infiltration is observed in various types of cancer including epithelial ovarian cancer. In addition, macrophages are involved in the formation of spheroids in the malignant ascites of ovarian cancer and promote cancer growth. iPS-ML, macrophage-like myelomonocytic cells generated from human induced pluripotent stem (iPS) cells, made close contacts with ovarian cancer cells in vitro. We hypothesized that, if we inoculate iPS-ML-producing IFN-β (iPS-ML/IFN-β) into the peritoneal cavity of patients with ovarian cancer, IFN-β produced by the iPS-ML/IFN-β would efficiently act on the cancer cells to suppress cancer growth. To evaluate this hypothesis, we injected iPS-ML/IFN-β into SCID mice bearing peritoneally disseminated human ovarian cancer cells, SKOV3. Immunohistochemical analysis of the intraperitoneal tumors detected iPS-ML/IFN-β infiltrating into the cancer tissues. Therapy with iPS-ML/IFN-β significantly suppressed tumor progression. In addition, dramatic reduction of cancer-related ascites was observed. Collectively, it is suggested that iPS-ML/IFN-β therapy offers a new approach for the treatment of patients with advanced ovarian cancer.

Synovitis of the affected joint is a common in avascular osteonecrosis (AVN). Increased levels of pro-inflammatory cytokine interleukin-6 (IL-6) have been reported in AVN, but the mechanism of this increase remains unclear. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, inhibits the release of inflammatory cytokines. Interferon β (IFN-β) has clear anti-inflammatory properties. We sought to investigate the effects of IFN-β treatment on AVN and to evaluate the specific signal pathway relating to IL-6 and SIRT1 affected during AVN. Using a dissection microscope, AVN was surgically induced in the distal femurs of mice. Exogenous IFN-β was administered to the model mice. The effects of exogenous IFN-β on AVN model mice were assessed using hematoxylin eosin and safranin-O staining, and bone resorption activity was measured using tartrate-resistant acid phosphatase (TRAP) and CD68 staining. Western blots, real-time RT-PCR, and immunohistochemical staining were performed to evaluate the production of SIRT1 and IL-6 in tissues. The RAW 264.7 cell line and bone marrow derived osteoclasts treated with exogenous IFN-β. Histological findings indicated well preserved trabecular bone and decreased osteoclast bone resorption activity in IFN-β treated mice compared with mice in the AVN group. Treatment with IFN-β increased SIRT1 expression and inhibited secretion of IL-6 in this AVN mouse model. IFN-β decreased IL-6 secretion by activating SIRT1 in the RAW 264.7 cell and bone marrow derived osteoclasts. Our work suggests that IFN-β could be used to treat AVN and that both SIRT1 and IL-6 are useful targets for treating patients with AVN.

Alendronate is commonly used for the treatment of postmenopausal osteoporosis; however, the underlying pathological molecular mechanisms of its action remain unclear. In the present study, the alendronate-treated signaling pathway in bone metabolism in rats with ovariectomy induced by osteoporosis was investigated. Rats with osteoporosis were orally administered alendronate or phosphate-buffered saline (control). In addition, the interferon-β (IFN-β)/signal transducer and activator of transcription 1 (STAT1) signaling pathway was investigated in osteoblasts following treatment with alendronate in vitro and in vivo. During the differentiation period, IFN-β (100 ng/ml) was used to treat the osteoblast cells, and the activity, viability and bone metabolism-associated gene expression levels (STAT1, p-STAT1, Fra1, TRAF6 and SOCS1) were analyzed in osteoblast cells. Histopathological changes were used to evaluate osteoblasts, osteoclasts, inflammatory phase of bone healing and osteonecrotic areas. The results demonstrated that alendronate significantly inhibited the activity of osteoporotic osteoclasts by stimulating expression of IFN-β, as well as markedly improved the viability and activity of osteoblasts compared with the control group. In addition, alendronate increased the expression and phosphorylation levels of STAT1 in osteoclasts, enhanced osteoblast differentiation, upregulated the expression levels of alkaline phosphatase and osteocalcin, and increased the expression of osteoblast differentiation-associated genes (osteocalcin, osterix and Runx2). Inhibition of IFN-β expression canceled the benefits of alendronate-mediated osteoblast differentiation. Notably, alendronate enhanced bone formation in rats with osteoporosis induced by ovariectomy. In conclusion, these findings suggest that alendronate can regulate osteoblast differentiation and bone formation in rats with osteoporosis induced by ovariectomy through upregulation of IFN-β/STAT1 signaling pathway.

Marek's disease virus (MDV) is an α-herpesvirus that causes immune suppression and T lymphoma in chickens. Toll-like receptor 3 (TLR3) is critical for the host immune response against MDV infection. Previously, our team demonstrated that pre-treatment of TLR3 agonist poly (I:C) inhibited Marek's disease virus infection in chicken embryo fibroblasts (CEFs). However, whether TLR3 inhibits the aggravation of MDV infection is unknown. In the current study, we found that TLR3 activation in MDV-infected CEFs effectively inhibited virus spread. Using pharmacological approaches, we revealed that pro-inflammatory cytokines and interferon-β induced by TLR3 could restrict Marek's disease virus infection. This study contributes to elucidating the function and mechanism of the TLR3 pathway in host immune responses against MDV infection.

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon‐β (IFNB1) induction. Using a comprehensive microscopy‐based high‐content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A‐interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF‐κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown—a hub protein highly targeted by several viruses—decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus‐mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF‐κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad‐spectrum antiviral therapies., Hepatitis C virus (HCV) hijacks the cellular host machinery to promote replication and to evade immune response. Using a microscopy‐based High Content Screening (HCS) assay, we demonstrated that HCV‐host interactors involved in nuclear transport are crucial for IRF3 and NFκB‐p65 signaling of IFNB1 response. To subvert innate immunity, NS3/4A interacts and cleaves IMPβ1, a key nucleocytoplasmic transport receptor, to prevent nuclear translocation of IRF3 and NFκB‐p65 transcription factors. Future studies will confirm if IMPβ1 is a prime target for several viruses to evade host defense.

Chlamydia trachomatis, an obligate intracellular pathogen, is the most common cause of bacterial sexually transmitted diseases, and it is potentially responsible for severe chronic sequelae, such as reactive arthritis. To date, details of the mechanisms by which Chlamydiae induce innate antimicrobial pathways in synovial fibroblasts, are not well characterized, therefore, herein, we investigated the effects of interferon (IFN)&alpha, IFN&beta, and IFN&gamma, on the infection, and replication phases of the C. trachomatis developmental cycle, as well as on the induction of pattern recognition receptors (PRRs) and IFN-related pathways. To do so, we set up an in vitro chlamydial-infection model of primary human synovial cells treated with IFNs before or after the infection. We then determined the number of chlamydial inclusion forming units and inclusion size, as well as the expression of toll like receptor (TLR)2, TLR3, TLR4, cyclic GMP-AMP synthase (cGAS), stimulator of IFN gene (STING), IRF9, ISG56, and GBP1. The main result of our study is the significant inhibition of C. trachomatis infection and replication in human synovial cells following the treatment with IFN&gamma, whereas IFN-I proved to be ineffective. Furthermore, IFN&gamma, greatly upregulated all the PRRs and ISGs examined. In conclusion, IFN&gamma, exhibited a potent anti-Chlamydia activity in human synovial cells as well as the ability to induce a strong increase of innate immune pathways.

Although genome-wide association studies have identified a number of common variants associated with multiple sclerosis (MS) susceptibility, little is known about the relevance of rare variants. Here, we aimed to explore the role of rare variants in 14 MS risk genes (FCRL1, RGS1, TIMMDC1, HHEX, CXCR5, LTBR, TSFM, GALC, TRAF3, STAT3, TNFSF14, IFI30, CD40, and CYP24A1) by targeted resequencing in an Iberian population of 524 MS cases and 546 healthy controls. Four rare variants-enriched regions within CYP24A1, FCRL1, RGS1, and TRAF3 were identified as significantly associated with MS. Functional studies revealed significantly decreased regulator of G protein signaling 1 (RGS1) gene expression levels in peripheral blood mononuclear cells from MS patients with RGS1 rare variants compared to noncarriers, whereas no significant differences in gene expression were observed for CYP24A1, FCRL1, and TRAF3 between rare variants carriers and noncarriers. Immunophenotyping showed significant decrease in RGS1 expression in peripheral blood B lymphocytes from MS patients with RGS1 rare variants relative to noncarriers. Lastly, peripheral blood mononuclear cell from MS patients carrying RGS1 rare variants showed significantly lower induction of RGS1 gene expression by interferon-β compared to MS patients lacking RGS1 variants. The presence of rare variants in RGS1 reinforce the ideas of high genetic heterogeneity and a role of rare variants in MS pathogenesis., This work was supported by grants from the Fondo de Investigación Sanitaria (FIS; grant number PI12/02229; Ministry of Science and Innovation, Spain), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarollo Regional (FEDER; grant number BFU2016‐77961‐P), Direcció General de Recerca ‐ Generalitat de Catalunya (2017‐SGR‐00702), Unidad de Excelencia María de Maeztu (MINECO; MDM‐2014‐0370), and Vall d'Hebrón Institut de Recerca (predoctoral grants program 2014).

Multiple sclerosis (MS) is characterized by peripheral and central inflammatory features, as well as demyelination and neurodegeneration. The available Food and Drug Administration (FDA)-approved drugs for MS have been designed to suppress the peripheral immune system. In addition, however, the effects of these drugs may be partially attributed to their influence on glial cells and neurons of the central nervous system (CNS). We here describe the molecular effects of the traditional and more recent FDA-approved MS drugs Fingolimod, Dimethyl Fumarate, Glatiramer Acetate, Interferon-β, Teriflunomide, Laquinimod, Natalizumab, Alemtuzumab and Ocrelizumab on microglia, astrocytes, neurons and oligodendrocytes. Furthermore, we point to a possible common molecular effect of these drugs, namely a key role for NFκB signaling, causing a switch from pro-inflammatory microglia and astrocytes to anti-inflammatory phenotypes of these CNS cell types that recently emerged as central players in MS pathogenesis. This notion argues for the need to further explore the molecular mechanisms underlying MS drug action.

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Cyclic di-AMP (c-di-AMP) is an important signaling molecule for pneumococci, and as a uniquely prokaryotic product it can be recognized by mammalian cells as a danger signal that triggers innate immunity. Roles of c-di-AMP in directing host responses during pneumococcal infection are only beginning to be defined. We hypothesized that pneumococci with defective c-di-AMP catabolism due to phosphodiesterase deletions could illuminate roles of c-di-AMP in mediating host responses to pneumococcal infection. Pneumococci deficient in phosphodiesterase 2 (Pde2) stimulated a rapid induction of interferon β (IFNβ) expression that was exaggerated in comparison to that induced by wild type (WT) bacteria or bacteria deficient in phosphodiesterase 1. This IFNβ burst was elicited in mouse and human macrophage-like cell lines as well as in primary alveolar macrophages collected from mice with pneumococcal pneumonia. Macrophage hyperactivation by Pde2-deficient pneumococci led to rapid cell death. STING and cGAS were essential for the excessive IFNβ induction, which also required phagocytosis of bacteria and triggered the phosphorylation of IRF3 and IRF7 transcription factors. The select effects of Pde2 deletion were products of a unique role of this enzyme in c-di-AMP catabolism when pneumococci were grown on solid substrate conditions designed to enhance virulence. Because pneumococci with elevated c-di-AMP drive aberrant innate immune responses from macrophages involving hyperactivation of STING, excessive IFNβ expression, and rapid cytotoxicity, we surmise that c-di-AMP is pivotal for directing innate immunity and host-pathogen interactions during pneumococcal pneumonia.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in cancer cells by binding to its receptors, death receptor 4 (DR4) and DR5, without affecting normal cells, and is therefore considered to be a promising antitumor agent for use in cancer treatment. However, several studies have indicated that most glioma cell lines display resistance to TRAIL-induced apoptosis. To overcome such resistance and to improve the efficacy of TRAIL-based therapies, identification of ideal agents for combinational treatment is important for achieving rational clinical treatment in glioblastoma patients. The main aim of this study was to investigate whether interferon-β (IFN-β) (with its pleiotropic antitumor activities) could sensitize malignant glioma cells to TRAIL-induced apoptosis using glioma cell lines. TRAIL exhibited a dose-dependent antitumor effect in all of the 7 types of malignant glioma cell lines, although the intensity of the effect varied among the cell lines. In addition, combined treatment with TRAIL (low clinical dose: 1 ng/ml) and IFN-β (clinically relevant concentration: 10 IU/ml) in A-172, AM-38, T98G, U-138MG and U-251MG demonstrated a more marked antitumor effect than TRAIL alone. Furthermore, the antitumor effect of the combined treatment with TRAIL and IFN-β may be enhanced via an extrinsic apoptotic system, and upregulation of DR5 was revealed to play an important role in this process in U-138MG cells. These findings provide an experimental basis to suggest that combined treatment with TRAIL and IFN-β may offer a new therapeutic strategy for malignant gliomas.

Background We previously reported that Vδ2+Vγ9+ γδ T cells were significantly decreased in multiple sclerosis (MS) patients without disease-modifying therapies (untreated MS) and were negatively correlated with Expanded Disability Status Scale (EDSS) scores, suggesting protective roles of Vδ2+Vγ9+ γδ T cells. Interferon-β (IFN-β) is one of the first-line disease-modifying drugs for MS. However, no previous studies have reported changes in γδ T cell subsets under IFN-β treatment. Therefore, we aimed to clarify the effects of the long-term usage of IFN-β on γδ T cell subsets in MS patients. Methods Comprehensive flow cytometric immunophenotyping was performed in 35 untreated MS and 21 MS patients on IFN-β for more than 2 years (IFN-β-treated MS) including eight super-responders fulfilling no evidence of disease activity criteria, and 44 healthy controls (HCs). Results The percentages of Vδ2+Vγ9+ cells in γδ T cells were significantly lower in untreated and IFN-β-treated MS patients than in HCs. By contrast, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were markedly higher in IFN-β-treated MS patients than in HCs and untreated MS patients (both p < 0.001). A significant negative correlation between the percentages of Vδ2+Vγ9+ cells in γδ T cells and EDSS scores was confirmed in untreated MS but not evident in IFN-β-treated MS. Moreover, class-switched memory B cells were decreased in IFN-β-treated MS compared with HCs (p < 0.001) and untreated MS patients (p = 0.006). Interestingly, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were negatively correlated with class-switched memory B cell percentages in all MS patients (r = − 0.369, p = 0.005), and the percentages of Vδ1−Vδ2−Vγ9− cells in Vδ1−Vδ2− γδ T cells were negatively correlated with EDSS scores only in IFN-β super-responders (r = − 0.976, p < 0.001). Conclusions The present study suggests that long-term usage of IFN-β increases Vδ1−Vδ2−Vγ9− γδ T cells, which are associated with a better outcome, especially in IFN-β super-responders. Thus, increased Vδ1−Vδ2−Vγ9− cells together with decreased class-switched memory B cells may contribute to the suppression of disease activity in MS patients under IFN-β treatment.

Background The airway epithelium is a major target tissue in respiratory infections, and its antiviral response is mainly orchestrated by the interferon regulatory factor-3 (IRF3), which subsequently induces type I (β) and III (λ) interferon (IFN) signalling. Dual specificity mitogen-activated protein kinase kinase (MEK) pathway contributes to epithelial defence, but its role in the regulation of IFN response in human primary airway epithelial cells (AECs) is not fully understood. Here, we studied the impact of a small-molecule inhibitor (MEKi) on the IFN response following challenge with two major respiratory viruses rhinovirus (RV2) and respiratory syncytial virus (RSVA2) and a TLR3 agonist, poly(I:C). Methods The impact of MEKi on viral load and IFN response was evaluated in primary AECs with or without a neutralising antibody against IFN-β. Quantification of viral load was determined by live virus assay and absolute quantification using qRT-PCR. Secretion of cytokines was determined by AlphaLISA/ELISA and expression of interferon-stimulated genes (ISGs) was examined by qRT-PCR and immunoblotting. A poly(I:C) model was also used to further understand the molecular mechanism by which MEK controls IFN response. AlphaLISA, siRNA-interference, immunoblotting, and confocal microscopy was used to investigate the effect of MEKi on IRF3 activation and signalling. The impact of MEKi on ERK and AKT signalling was evaluated by immunoblotting and AlphaLISA. Results Here, we report that pharmacological inhibition of MEK pathway augments IRF3-driven type I and III IFN response in primary human AECs. MEKi induced activation of PI3K-AKT pathway, which was associated with phosphorylation/inactivation of the translational repressor 4E-BP1 and activation of the protein synthesis regulator p70 S6 kinase, two critical translational effectors. Elevated IFN-β response due to MEKi was also attributed to decreased STAT3 activation, which consequently dampened expression of the transcriptional repressor of IFNB1 gene, PRDI-BF1. Augmented IFN response translated into inhibition of rhinovirus 2 replication in primary AECs but not respiratory syncytial virus A2. Conclusions Our findings unveil MEK as a key molecular mechanism by which rhinovirus dampens the epithelial cell’s antiviral response. Our study provides a better understanding of the role of signalling pathways in shaping the antiviral response and suggests the use of MEK inhibitors in anti-viral therapy against RV. Electronic supplementary material The online version of this article (10.1186/s12964-019-0378-7) contains supplementary material, which is available to authorized users.