Type I Interferon Research Articles

Targeting SAMHD1 Promotes Anti-Tumor Immunity in Acute Myeloid Leukemia

The overall outcome of AML, which is characterized by HOXA9 overexpression (>50% cases), remains unfavorable. Success of allogeneic hematopoietic stem cell transplantation and immunotherapy highlights that T cell activation is critical for AML eradication. Notably, suppression of T cell immunity is common in the AML BM microenvironment, thus there is a pressing need to define underlying mechanisms. Recent studies reveal that the dNTP and Ara-CTP (active form of cytarabine) hydrolase SAMHD1, whose loss-of-function mutation is seen in inflammatory encephalopathy named Aicardi-Goutières syndrome, limits type-1 Interferon (IFN-I) and T cell responses by suppressing cGAS/STING signaling. Interestingly, our analysis of GEO datasets showed elevated SAMHD1 levels in specimens from AML patients relative to healthy donors, correlated with HOXA9 expression (A) and decreased T cell function. We thus asked whether SAMHD1 levels are associated with a particular cytogenetic change or mutation upstream of HOXA9 dysregulation. Accordingly, analysis of GSE data demonstrated that AML cases with MLL rearrangement (MLL-r, t[11q23]) or NPM1 mutation (NPM1c) exhibited elevated SAMHD1 levels relative to those without either alteration (B, C). Notably, HOXA9 knockdown (KD) decreased SAMHD1 protein levels in Molm13 (MLL-r+) and OCI-AML3 (NPM1c+) cells. ChIP-Q-PCR analysis of cord blood CD34+ cells engineered with Flag-tagged HoxA9 showed significant enrichment of HoxA9 protein at the predicted SAMHD1 promoter region relative to a control site. That region also showed significant enrichment of H3K27Ac (an active transcription mark) in HoxA9-overexpressing relative to control cells. We next assessed SAMHD1 loss-of-function in MLL-AF9 (MA9) knock-in mice, first by transducing leukemia cells from MA9+ BM with a doxycycline (DOX)-inducible control or shSAMHD1 and then transplanting them into either WT immunocompetent or immunodeficient (Rag2-/-) recipients to induce leukemia. Following DOX treatment, Rag2-/- mice bearing SAMHD1-KD or control transplants exhibited comparable survival. However, WT recipients bearing SAMHD1-KD AML survived significantly longer than controls (D, E). Moreover, SAMHD1-KD-mediated leukemia ablation was reproduced in HOXA9-Meis1 transduction/transplantation AML. Furthermore, depletion of T cells, but not B cells, blocked survival advantages seen in SAMHD1-KD-transplanted animals. To determine if IFN-I responses underlie these effects, we implanted SAMHD1-KD MA9 cells into WT or Ifnar1-/- recipients and observed significant abolition of SAMHD1-KD anti-AML effects. Moreover, SAMHD1-KD induced cGAS activation, as evidenced by elevated levels of the immune transmitter cGAMP in BM fluid from SAMHD1-KD relative to control MA9 mice. Accordingly, we observed upregulation of multiple interferon-stimulated genes (ISGs) such as ISG15 and CXCL10 downstream of cGAS/STING after SAMHD1-KD, effects blocked by cGAS deletion. To determine if immune activation requires SAMHD1 catalysis, we knocked out endogenous SAMHD1 in THP1 cells, which we then engineered cells to express doxycycline-inducible SAMHD1 variants including WT (SAMWT), catalytically-dead mutant SAMHD1 (SAMMT) or empty vector (MOCK). Comparison of ISGs expression in each group indicated that, relative to MOCK, SAMWT but not SAMMT downregulated ISGs, suggesting that blocking SAMHD1 catalysis promotes innate immunity. To identify SAMHD1 inhibitors, we conducted a structure-based virtual screen of 8,000,000 compounds (Molport). We then assessed the top 500 candidates using cell-based phenotypic screening based on cytarabine sensitization, namely, the inhibitor should sensitize SAMWT but not SAMMT cells to Ara-C toxicity (F). We then selected the 20 best candidates to evaluate for inhibition of SAMHD1 catalysis and chose Samin27 as the most potent lead compound. Importantly, we confirmed direct Samin27 interaction with SAMHD1 protein by nuclear magnetic resonance (NMR) (G). Samin27 treatment of SAMHD1-proficient human and mouse AML lines significantly upregulated ISGs expression. These results suggest that SAMHD1 plays an oncogenic role in AML by suppressing innate immunity. Further analysis is required to determine whether our compound can ablate AML in vivo when combined with currently available immune checkpoint inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CTIM-05. FINAL RESULTS OF 2-THE-TOP: A PILOT PHASE 2 STUDY OF TTFIELDS (OPTUNE) PLUS PEMBROLIZUMAB PLUS MAINTENANCE TEMOZOLOMIDE (TMZ) IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (NDGBM)

Abstract INTRODUCTION TTFields induce anti-tumor immunity via type-1 interferon (T1IFN) pathways of the STING and AIM2 inflammasomes. Thus, TTFields may synergize with immune checkpoint inhibitors to prolong survival in GBM. METHODS We enrolled 26 ndGBM patients in a pilot phase 2 study combining TTFields, pembrolizumab and maintenance TMZ. Primary endpoints were PFS versus case-matched controls of TTFields plus TMZ in the EF-14 study and immune signatures by multiomics of PBMCs and tumors. Secondary endpoints included toxicity and OS. RESULTS The median age was 60.5 years. Fourteen (54%) had biopsy only or partial resection. Nineteen (73%) had unmethylated MGMT and 3 (11.5%) had an IDH mutation. As of 2/16/22, 7 (27%) were progression-free and 14 (54%) were alive with median follow-up of 16.8 months. Median PFS was 12.1 months versus 7.9 months in a case-matched control cohort of 26 patients (HR = 0.456; 95% CI: 0.222-0.937; P = 0.015). Twelve-month PFS was 52.4% (95% CI: 31.4-69.6%) versus 20.4% (95% CI: 5.5-41.7%) in controls; P = 0.012. Six of 15 (40%) patients with measurable disease achieved partial or complete response. Median OS was 25.2 months versus 15.9 months in controls (HR = 0.382; 95% CI: 0.168-0.861; P = 0.078). Two-year OS was 57.8% (95% CI: 33.7-75.9%) versus 19.2% (95% CI: 7.0-36.0%) in controls; P = 0.002. In a Cox regression analysis adjusting for key prognostic factors, P-value reached 0.033 for PFS and 0.020 for OS. Molecular analyses confirmed robust T cell activation via the T1IFN trajectory, which highly correlated with TCRαβ clonal expansion (Spearman coefficient = -0.8; P = 0.014), and defined a T cell-based gene signature of TTFields effects. The most common serious adverse events were thromboses, seizures, and metabolic disturbances in 4 (15%), 3 (11.5%), and 2 (8%) patients, respectively. CONCLUSIONS The triple combination demonstrated acceptable toxicity and promising efficacy in ndGBM. Survival and molecular data will be updated.

Rift Valley Fever Virus Non-Structural Protein S Is Associated with Nuclear Translocation of Active Caspase-3 and Inclusion Body Formation.

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies.

Abstract 12645: Role of Viral Rna-Triggered Innate Immune Activation on Cardiac Arrhythmias Associated With Covid-19

Background: The COVID-19 pandemic has highlighted how little is known about how double stranded RNA (dsRNA) viruses like the SARS-CoV-2 impact cardiac physiology acutely and long-term. While it is unclear if the cardiac effects are direct or indirect, adverse effects in patients are common, including cardiac inflammation, electrophysiological changes in QT interval, atrial fibrillation, bradyarrhythmia, and ventricular tachycardia. Mechanisms underlying these COVID-19-associated arrhythmias are unknown. Methods: We assess the effects of SARS-CoV-2 infection on cardiac electrophysiology and inflammatory pathway activation in a hamster model over a 4-week timespan. We further test if activation of antiviral innate immune responses with a dsRNA mimetic, Poly I:C (PI:C) induces similar effects in uninfected hearts and in human iPSC-derived cardiomyocytes (hiPSC-CMs), examining the determinants of RNAseL activation, interferon (IFN) signaling, and cytokine release in vivo and in vitro. Results: Intranasal SARS-CoV-2 USA-WA1 infection of hamsters caused ECG abnormalities including bradycardia and cardiac conduction defects including sinus pauses, 2 nd and 3 rd degree Atrio-Ventricular block. Bradycardia peaked at 4-5 days after infection. Mean RR interval increased from 182±4ms pre- to 233±6ms post-infection, without significant increases in mock-infected hamsters. In the absence of viral infection, ventricular injection of PI:C induced similar arrhythmias and induction of IFN-stimulated genes. PI:C treatment of hiPSC-CMs induced robust increases in type-I IFN pathway proteins STAT1/pSTAT1, OAS2 (48% increase) and OAS3 (32% increase) and a robust cytokine response. Negative effects on excitation-contraction coupling after 72hr of PI:C treatment, manifested as a 25% reduction in hiPSC-CM Ca 2+ transient peaks. In vitro studies showed increased RNA degradation after PI:C, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. Conclusions: COVID-19 in the hamster model recapitulates a subset of arrhythmias observed in COVID-19 patients. Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, may contribute to SARS-CoV-2-associated cardiac dysfunction.

Inflammation and immune activation are associated with risk of Mycobacterium tuberculosis infection in BCG-vaccinated infants

Tuberculosis vaccine development is hindered by the lack of validated immune correlates of protection. Exploring immune correlates of risk of disease and/or infection in prospective samples can inform this field. We investigate whether previously identified immune correlates of risk of TB disease also associate with increased risk of M.tb infection in BCG-vaccinated South African infants, who became infected with M.tb during 2-3 years of follow-up. M.tb infection is defined by conversion to positive reactivity in the QuantiFERON test. We demonstrate that inflammation and immune activation are associated with risk of M.tb infection. Ag85A-specific IgG is elevated in infants that were subsequently infected with M.tb, and this is coupled with upregulated gene expression of immunoglobulin-associated genes and type-I interferon. Plasma levels of IFN-alpha2, TNF-alpha, CXCL10 (IP-10) and complement C2 are also higher in infants that were subsequently infected with M.tb.

Manganese induces tumor cell ferroptosis through type-I IFN dependent inhibition of mitochondrial dihydroorotate dehydrogenase.

Ferroptosis is a novel form of regulated cell death characterized by the iron-dependent accumulation of lipid peroxides to lethal levels, which is morphologically, biochemically, and genetically distinct from apoptosis, necroptosis, autophagy, and pyroptosis. Manganese play an important role in innate immunity and antitumor immunity. Many manganese-based nanomaterials induce tumor cell death by catalyzing the production of reactive oxygen species (ROS) within the tumor. However, the exact underlying mechanisms remain unclear. As research on ferroptosis advances and its regulatory mechanisms in tumors continue to be refined, more evidence has suggested that triggering ferroptosis in tumor cells is an effective strategy for tumor treatment. In this study, we found that administration of MnCl2 to tumor cells resulted in lipid peroxidation and increased the levels of mitochondrial ROS, consequently leading to ferroptosis. Dihydroorotate dehydrogenase (DHODH)-mediated ferroptosis defence is a targetable vulnerability in cancer. We show that MnCl2 downregulated DHODH expression in tumor cells, resulting in increased mitochondrial ROS and lipid peroxidation to induce ferroptosis. In addition, MnCl2 enhanced the phosphorylation levels of STING, TBK1, and IRF3 and upregulated the expression of type-I interferon (IFN), produced by the cGAS-STING signaling pathway. When inhibiting the cGAS-STING signaling pathway or type-I IFN, DHODH expression was restored, reversing lipid peroxidation and ROS production and rescuing MnCl2-induced ferroptosis.. Knockout of IFNAR1 or overexpression of DHODH weakens the antitumor effect of MnCl2. Mechanistically, these results revealed that Manganese treatment-activated cGAS-STING signaling promote mitochondrial lipid peroxidation and ROS production by releasing type-I IFNs that reduce DHODH function and thereby inducing ferroptosis in tumor cells. This may provide a new strategy to complement existing antitumor treatment regimens.

ODP571 Blockade of ErbB-2 Nuclear Function Induces the Interferon Signaling Pathway in Breast Cancer Models Resistant to Trastuzumab

Abstract ErbB-2, a member of ErbB family of receptor tyrosine kinases, is a key oncogenic driver in breast cancer. Despite clinical efficiency of ErbB-2-targeted therapies (trastuzumab, TZ), resistance to drugs is a major issue in the clinic. While ErbB-2 is mainly a plasma membrane-bound receptor, it also migrates to the nucleus (NErbB-2) where it can act as a transcription factor or coactivator. We previously reported that NErbB-2 is a major proliferation driver in TZ-resistant breast cancer. To investigate the NErbB-2 dependent transcriptome, RNAseq was performed using a TZ-resistant breast cancer model (JIMT-1 cells) with high constitutive levels of NErbB-2. JIMT-1 cells were transfected with an ErbB-2 nuclear localization domain mutant (hErbB-2ΔNLS), which also acts as a dominant-negative inhibitor of endogenous NErbB-2 migration. Exclusion of ErbB-2 from the nucleus resulted in up-regulation of 280 genes and down-regulation of 33 genes. Functional analysis revealed that NErbB-2 blockade enriched the expression of genes involved in type-I interferon (IFN) signaling pathway. IFNB1 and its downstream effectors OAS2 and TRIM22 were among the top up-regulated genes. In an independent breast cancer model (i. e., HCC-1569 cells), exclusion of NErbB-2 from the nucleus also induced expression of these genes. Blockade of NErbB-2 localization by injection of the hErbB-2ΔNLS mutant into JIMT-1 tumor xenografts significantly inhibited in vivo tumor growth and induced mRNA expression of IFNB1, OAS2 and TRIM22. Interestingly, blockade of NErbB-2 localization by treatment with Retro-2, an inhibitor of the retrograde transport, showed similar effects consistent with modulation of the IFN signaling pathway by NErbB-2. Bioinformatic analyses showed that both the promoter and the coding region of the IFNB1 gene contain ErbB-2 associated sequences (HAS sites). ChIP-PCR analyses revealed ErbB-2 recruitment to the HAS sites of the IFNB1 promoter and coding regions in normal growth conditions. Transfection of JIMT-1 cells with the hErbB-2ΔNLS mutant abolished the recruitment of ErbB-2 at the IFNB1 gene and also caused an increase in histone H4 acetylation, a marker of active gene transcription. NErbB-2 immunostaining in a cohort of 32 primary invasive ErbB-2-positive breast carcinomas treated with TZ revealed that NErbB-2 expression correlated with a poor disease-free survival. While this cohort is small, the findings suggest that NErbB-2 could be used as a biomarker of poor response to TZ in the clinic. In summary, our findings indicate that NErbB-2 drives the growth of TZ-resistant breast cancer cells via transcriptional repression of the IFNB1 signaling pathway, and highlight NErbB-2 as a therapeutic target and biomarker in TZ-resistant breast cancer. Presentation: No date and time listed

H1N1 Influenza A Virus Protein NS2 Inhibits Innate Immune Response by Targeting IRF7.

Influenza A virus (IAV) is a globally distributed zoonotic pathogen and causes a highly infectious respiratory disease with high morbidity and mortality in humans and animals. IAV has evolved various strategies to counteract the innate immune response, using different viral proteins. However, the mechanisms are not fully elucidated. In this study, we demonstrated that the nonstructural protein 2 (NS2) of H1N1 IAV negatively regulate the induction of type-I interferon. Co-immunoprecipitation experiments revealed that NS2 specifically interacts with interferon regulatory factor 7 (IRF7). NS2 blocks the nuclear translocation of IRF7 by inhibiting the formation of IRF7 dimers, thereby prevents the activation of IRF7 and inhibits the production of interferon-beta. Taken together, these findings revealed a novel mechanism by which the NS2 of H1N1 IAV inhibits IRF7-mediated type-I interferon production.

Heterogeneous RNA editing and influence of ADAR2 on mesothelioma chemoresistance and the tumor microenvironment.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. We found that tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3'UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment, and type-1 interferon signaling upregulation, leading to changes in the microenvironment invivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also in chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

Genome-Wide Gene Expression Analysis Reveals Unique Genes Signatures of Epithelial Reorganization in Primary Airway Epithelium Induced by Type-I, -II and -III Interferons.

Biosensors such as toll-like receptors (TLR) induce the expression of interferons (IFNs) after viral infection that are critical to the first step in cell-intrinsic host defense mechanisms. Their differential influence on epithelial integrity genes, however, remains elusive. A genome-wide gene expression biosensor chip for gene expression sensing was used to examine the effects of type-I, -II, and -III IFN stimulation on the epithelial expression profiles of primary organotypic 3D air-liquid interface airway cultures. All types of IFNs induced similar interferon-stimulated genes (ISGs): OAS1, OAS2, and IFIT2. However, they differentially induced transcription factors, epithelial modulators, and pro-inflammatory genes. Type-I IFN-induced genes were associated with cell-cell adhesion and tight junctions, while type-III IFNs promoted genes important for transepithelial transport. In contrast, type-II IFN stimulated proliferation-triggering genes associated and enhanced pro-inflammatory mediator secretion. In conclusion, with our microarray system, we provide evidence that the three IFN types exceed their antiviral ISG-response by inducing distinct remodeling processes, thereby likely strengthening the epithelial airway barrier by enhancing cross-cell-integrity (I), transepithelial transport (III) and finally reconstruction through proliferation (II).

Interferon-Driven Immune Dysregulation in Common Variable Immunodeficiency–Associated Villous Atrophy and Norovirus Infection

PurposeAbout 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute.MethodsDuodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest.ResultsVA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples.ConclusionsOur study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.Graphical abstract

Cancer immunotherapy strategies that target the cGAS-STING pathway.

Activation of the cGAS-STING pathway by cytoplasmic DNA induces the production of Type-1 interferons. Recent advances in research suggest that the cGAS-STING pathway is involved in different parts of the cancer-immunity cycle (CIC) to promote or suppress antitumor immune responses. Combination therapy of STING agonists has made certain progress in preclinical as well as clinical trials, but the selection of combination therapy regimens remains a challenge. In this review, we summarize the role of the cGAS-STING in all aspects of CIC, and focus on the combination immunotherapy strategies of STING agonists and current unsolved challenges.

Stochastic dynamics of Type-I interferon responses.

Interferon (IFN) activates the transcription of several hundred of IFN stimulated genes (ISGs) that constitute a highly effective antiviral defense program. Cell-to-cell variability in the induction of ISGs is well documented, but its source and effects are not completely understood. The molecular mechanisms behind this heterogeneity have been related to randomness in molecular events taking place during the JAK-STAT signaling pathway. Here, we study the sources of variability in the induction of the IFN-alpha response by using MxA and IFIT1 activation as read-out. To this end, we integrate time-resolved flow cytometry data and stochastic modeling of the JAK-STAT signaling pathway. The complexity of the IFN response was matched by fitting probability distributions to time-course flow cytometry snapshots. Both, experimental data and simulations confirmed that the MxA and IFIT1 induction circuits generate graded responses rather than all-or-none responses. Subsequently, we quantify the size of the intrinsic variability at different steps in the pathway. We found that stochastic effects are transiently strong during the ligand-receptor activation steps and the formation of the ISGF3 complex, but negligible for the final induction of the studied ISGs. We conclude that the JAK-STAT signaling pathway is a robust biological circuit that efficiently transmits information under stochastic environments.

Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN–γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN–γ is best described in humans, but less of IFN–γ in fish species has been described at protein level. In the present study, IFN–γ from Gadus macrocephalus (GmIFN–γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN–γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN–γ signature sequence at the C-terminal. GmIFN–γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN–γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN–γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET–32a–GmIFN–γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA–GmIFN–γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN–γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1–GmIFN–γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN–γ protein could also induce MICA expression, indicating that GmIFN–γ could stimulate immune response. The yeast GS115 with GmIFN–γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN–γ was upregulated significantly; however, genes of the interferon type–I signal pathway were not well stimulated.

Galectin-9 protects humanized-ACE2 immunocompetent mice from SARS-CoV-2 infection.

SARS-CoV-2 remains a global health crisis even with effective vaccines and the availability of FDA approved therapies. Efforts to understand the complex disease pathology and develop effective strategies to limit mortality and morbidity are needed. Recent studies reveal circulating Galectin-9 (gal-9), a soluble beta-galactoside binding lectin with immunoregulatory properties, are elevated in SARS-CoV-2 infected individuals with moderate to severe disease. Moreover, in silico studies demonstrate gal-9 can potentially competitively bind the ACE2 receptor on susceptible host cells. Here, we determined whether early introduction of exogenous gal-9 following SARS-CoV-2 infection in humanized ACE2 transgenic mice (K18-hACE2) may reduce disease severity. Mice were infected and treated with a single dose of a human recombinant form of gal-9 (rh-gal-9) and monitored for morbidity. Subgroups of mice were humanely euthanized at 2- and 5- days post infection (dpi) for viral levels by plaque assay, immune changes measures by flow cytometry, and soluble mediators by protein analysis from lung tissue and bronchoalveolar Lavage fluid (BALF). Mice treated with rh-gal-9 during acute infection had improved survival compared to PBS treated controls. At 5 dpi, rh-gal-9 treated mice had enhanced viral clearance in the BALF, but not in the lung parenchyma. Increased T and dendritic cells and decreased neutrophil frequencies in the lung at 5 dpi were observed, whereas BALF had elevated levels of type-I interferons and proinflammatory cytokines. These results suggest a role for rh-gal-9 in limiting acute COVID-19. Further studies are required to determine the optimal design of gal-9 treatment to effectively ameliorate COVID-19 disease.

MBTPS1 regulates proliferation of colorectal cancer primarily through its action on sterol regulatory element-binding proteins.

Among the main metabolic pathways implicated in cancer cell proliferation are those of cholesterol and fatty acid synthesis, both of which are tightly regulated by sterol regulatory element-binding proteins (SREBPs). SREBPs are activated through specific cleavage by membrane-bound transcription factor protease 1 (MBTPS1), a serine protease that cleaves additional substrates (ATF6, BDNF, CREBs and somatostatin), some of which are also implicated in cell proliferation. The goal of this study was to determine whether MBTPS1 may serve as a master regulator in proliferation of colorectal cancer (CRC). Tumors from CRC patients showed variable levels of MBTPS1 mRNA, which were in positive correlation with the levels of SREBPs and ATF6, and in reverse correlation with BDNF levels. Chemical inhibition of MBTPS1 activity in two CRC-derived cell lines resulted in a marked decrease in the levels of SREBPs, but not of its other substrates and a marked decrease in cell proliferation, which suggested that MBTPS1 activity is critical for proliferation of these cells. In accordance, CRISPR/Cas9 targeted knockout (KO) of the MBTPS1 gene resulted in the survival of only a single clone that presented a phenotype of severely attenuated proliferation and marked downregulation of several energy metabolism pathways. We further showed that survival of the MBTPS1 KO clone was dependent upon significant upregulation of the type-1 interferon pathway, the inhibition of which halted proliferation entirely. Finally, rescue of the MBTPS1 KO cells, resulted in partial restoration of MBTPS1 levels, which was in accordance with partial recovery in proliferation and in SREBP levels. These finding suggest that MBTPS1 plays a critical role in regulating colon cancer proliferation primarily through SREBP-associated lipid metabolism, and as such may serve as a possible therapeutic target in CRC.

Multifunctional PVCL nanogels enable magnetic resonance imaging and immunostimulated radiotherapy of orthotopic glioblastoma

Despite radiotherapy (RT) is considered as an indispensable treatment that improves clinical symptoms and survival rates of glioblastoma multiforme (GBM), the overall survival time of GBM based on multimodal treatment synergic with RT is still unsatisfying. Herein, we designed an immunostimulated nanoplatform consisting of MnO2 and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) within the poly(N-vinylcaprolactam) nanogels (PVCL-MnO2-CpG NGs) for magnetic resonance imaging (MRI)-guided immuno-activated RT against GBM. In this system, I) CpG ODNs can be protected by PVCL NGs from degradation, which can increase the cellular uptake by immune cells and promote the activation of Toll-like receptor 9 (TLR9) pathway, thus enhance the immune responses to assist the therapeutic efficacy of tumour RT. II) PVCL-MnO2-CpG NGs are able to be dissociated to release Mn2+ in the tumour microenvironment, resulting in excellent T1-weighted tumour MRI along with the activation of cyclic GMP–AMP synthase/stimulator of interferon genes (cGAS/STING) pathway and elevated secretion of type-I interferons (IFN-I) in the downstream. III) MnO2 can also act as nanoenzyme to relieve tumour radioresistance through increasing the reactive oxygen species (ROS) level. Compared with RT alone, combinational immunoradiotherapy remarkably promotes the proportion of cytotoxic T cells (CD3+CD8+) and exhibits systemic therapeutic efficacy. Thus, MnO2 and CpG ODNs-loaded PVCL NGs may serve as a potential theranostic platform for MRI-guided immunostimulated RT of GBM.

Hycanthone Inhibits Inflammasome Activation and Neuroinflammation-Induced Depression-Like Behaviors in Mice.

Despite the various medications used in clinics, the efforts to develop more effective treatments for depression continue to increase in the past decades mainly because of the treatment-resistant population, and the testing of several hypotheses- and target-based treatments. Undesirable side effects and unresponsiveness to current medications fuel the drive to solve this top global health problem. In this study, we focused on neuroinflammatory response-mediated depression which represents a cluster of depression etiology both in animal models and humans. Several meta-analyses reported that proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were increased in major depressive disorder patients. Inflammatory mediators implicated in depression include type-I interferon and inflammasome pathways. To elucidate the molecular mechanisms of neuroinflammatory cascades underlying the pathophysiology of depression, we introduced hycanthone, an antischistosomal drug, to check whether it can counteract depressive-like behaviors in vivo and normalize the inflammation-induced changes in vitro. Lipopolysaccharide (LPS) treatment increased proinflammatory cytokine expression in the murine microglial cells as well as the stimulation of type I interferon-related pathways that are directly or indirectly regulated by Janus kinase-signal transducer and activator of transcription (JAK-STAT) activation. Hycanthone treatment attenuated those changes possibly by inhibiting the JAK-STAT pathway and inflammasome activation. Hycanthone also ameliorated depressive-like behaviors by LPS. Taken together, we suggest that the inhibitory action of hycanthone against the interferon pathway leading to attenuation of depressive-like behaviors can be a novel therapeutic mechanism for treating depression.

MITA/STING-mediated antiviral immunity and autoimmunity: the evolution, mechanism, and intervention.

Mediator of IRF3 activation (MITA, also known as stimulator of interferon genes (STING) and endoplasmic reticulum interferon stimulator (ERIS)) is an ER-associated protein that senses cellular and bacterium-derived cyclic dinucleotide (CDN), leading to induction of type-I interferons (IFNs) and innate immune responses against viruses and bacteria. Recently, it has become clear that sensing of CDN and induction of autophagy are two evolutionarily conserved functions of MITA, predating its role in mediating type-I IFN induction. Studies have shown that MITA-mediated signaling promotes a number of autoimmune disorders caused by gene mutations in human. Here, we summarize the most recent progress on MITA-mediated signaling in a view of evolution and highlight the roles of MITA in human inflammatory disorders caused by gene mutations and in genetically modified mouse models. We also briefly introduce the chemicals targeting MITA and discuss their potential in treatment of MITA-mediated inflammatory diseases. Finally, we propose several key questions that should be addressed for targeting MITA for treatment of related autoimmune diseases.

Regulation of innate immunity by Nrf2.

The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) has been mainly investigated as a regulator of redox homeostasis. However, research over the past years has implicated Nrf2 as an important regulator of innate immunity. Here, we discuss the role of Nrf2 in the innate immune response, highlighting the interaction between Nrf2 and major components of the innate immune system. Indeed, Nrf2 has been shown to widely control the immune response by interacting directly or indirectly with important innate immune components, including the toll-like receptors-Nuclear factor kappa B (NF-kB) pathway, inflammasome signaling, and the type-I interferon response. This indicates an essential role for Nrf2 in diseases related to microbial infections, inflammation, and cancer. Yet, further studies are required to determine the exact mechanism underpinning the interactions between Nrf2 and innate immune players in order to allow a better understanding of these diseases and leverage new therapeutic strategies.