Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.

STINGing away the pain: the role of interferon-stimulated genes.

Pain and inflammation are biologically intertwined responses that warn the body of potential danger. In this issue of the JCI, Defaye, Bradaia, and colleagues identified a functional link between inflammation and pain, demonstrating that inflammation-induced activation of stimulator of IFN genes (STING) in dorsal root ganglia nociceptors reduced pain-like behaviors in a rodent model of inflammatory pain. Utilizing mice with a gain-of-function STING mutation, Defaye, Bradaia, and colleagues identified type I IFN regulation of voltage-gated potassium channels as the mechanism of this pain relief. Further investigation into mechanisms by which proinflammatory pathways can reduce pain may reveal druggable targets and insights into new approaches for treating persistent pain.

Everlasting first impressions in B cells.

Initial imprinting by type 1 interferons shapes memory B cell generation in chronic viral infection.

The alteration of NK cells phenotypes related to the functions and dengue disease outcomes.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease.

ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is a challenging disease, especially for patients with immunologically "cold" tumors devoid of tumor-infiltrating lymphocytes (TILs). We found that HGSC exhibits among the highest levels of MYCN expression and transcriptional signature across human cancers, which is strongly linked to diminished features of antitumor immunity. N-MYC repressed basal and induced IFN type I signaling in HGSC cell lines, leading to decreased chemokine expression and T cell chemoattraction. N-MYC inhibited the induction of IFN type I by suppressing tumor cell-intrinsic STING signaling via reduced STING oligomerization, and by blunting RIG-I-like receptor signaling through inhibition of MAVS aggregation and localization in the mitochondria. Single-cell RNA sequencing of human clinical HGSC samples revealed a strong negative association between cancer cell-intrinsic MYCN transcriptional program and type I IFN signaling. Thus, N-MYC inhibits tumor cell-intrinsic innate immune signaling in HGSC, making it a compelling target for immunotherapy of cold tumors.

Colorectal cancer-specific IFNß delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy.

BACKGROUND: Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING signaling and increases cancer immunogenicity and enhances the antitumor immune response to increase therapeutic efficacy. However, cGAS/STING deficiency in colorectal cancer (CRC) may suppress the RT-induced antitumor immunity. Therefore, we aimed to evaluate the importance of the dsRNA-mediated antitumor immune response induced by RT in patients with CRC. METHODS: Cytosolic dsRNA level and its sensors were evaluated via cell-based assays (co-culture assay, confocal microscopy, pharmacological inhibition and immunofluorescent staining) and in vivo experiments. Biopsies and surgical tissues from patients with CRC who received preoperative chemoradiotherapy (neoCRT) were collected for multiplex cytokine assays, immunohistochemical analysis and SNP genotyping. We also generated a cancer-specific adenovirus-associated virus (AAV)-IFNß1 construct to evaluate its therapeutic efficacy in combination with RT, and the immune profiles were analyzed by flow cytometry and RNA-seq. RESULTS: Our studies revealed that RT stimulates the autonomous release of dsRNA from cancer cells to activate TLR3-mediated IFN-I signatures to facilitate antitumor immune responses. Patients harboring a dysfunctional TLR3 variant had reduced serum levels of IFN-I-related cytokines and intratumoral CD8+ immune cells and shorter disease-free survival following neoCRT treatment. The engineered cancer-targeted construct AAV-IFNß1 significantly improved the response to RT, leading to systematic eradication of distant tumors and prolonged survival in defective TLR3 preclinical models. CONCLUSION: Our results support that increasing cancer-intrinsic IFNß1 expression is an immunotherapeutic strategy that enhances the RT-induced antitumor immune response in locally patients with advanced CRC with dysfunctional TLR3.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Mitochondrial DNA release and sensing in innate immune responses.

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

The regulation of cGAS-STING signaling by RNA virus-derived components.

The Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) serves as a key innate immune signaling axis involved in the regulation of various human diseases. It has been found that cGAS-STING pathway can recognize a variety of cytosolic double-stranded DNA (dsDNA), contributing to cause a robust type I interferon response thereby affecting the occurrence and progression of viral infection. Accumulating evidence indicates RNA virus-derived components play an important role in regulating cGAS-STING signaling, either as protective or pathogenic factors in the pathogenesis of diseases. Thus, a comprehensive understanding of the function of RNA virus-derived components in regulating cGAS-STING signaling will provide insights into developing novel therapies. Here, we review the existing literature on cGAS-STING pathway regulated by RNA virus-derived components to propose insights into pharmacologic strategies targeting the cGAS-STING pathway.

Ionizing Radiation-treated NRF2-deficient Cancer Cells Promote Type I Interferon Signaling in Immune Cells.

BACKGROUND/AIM: Nuclear factor erythroid-derived 2-related factor-2 (NRF2) is a transcription factor that regulates stress response genes. It negatively regulates the immune system by acting as a transcriptional repressor of inflammatory genes or suppressing type I interferon (IFN) production pathways. NRF2 is often over-expressed in some tumors, including non-small cell lung cancer, and modulates these tumors via an immune-cold microenvironment. Thus, strategies to convert cold tumors into hot tumors are effective for cancer treatment. MATERIALS AND METHODS: NRF2 was knocked-down or over-expressed in human cancer cells (A549, HeLa, H1299, H1650) and mouse mammary adenocarcinoma TS/A cells. Cells were irradiated or transfected with poly(I:C), and changes in type I IFN levels were examined using quantitative real-time polymerase chain reaction and western blotting. Cytosolic DNA was assayed via PicoGreen staining and immune and cancer cells were co-cultured. RESULTS: Regulation of NRF2 expression altered type I IFN levels in the human lung cancer cell line A549 and several solid tumors. Down-regulation of NRF2 resulted in increased levels of cytosolic DNA and activated the cGAS-STING pathway. We confirmed that type I IFN was induced in NRF2-down-regulated tumor cells using ionizing radiation (IR). Furthermore, when dendritic cells and macrophages were co-cultured with IR-exposed NRF2 knockdown tumor cells, the immune cells produced more IFNB1 and CXCL10. CONCLUSION: The immunosuppressive tumor cell environment is improved by NRF2 down-regulation, and IR treatment may promote immune cell signaling activation.

The association between single-nucleotide polymorphisms within type 1 interferon pathway genes and human immunodeficiency virus type 1 viral load in antiretroviral-naïve participants.

BACKGROUND: Human genetic contribution to HIV progression remains inadequately explained. The type 1 interferon (IFN) pathway is important for host control of HIV and variation in type 1 IFN genes may contribute to disease progression. This study assessed the impact of variations at the gene and pathway level of type 1 IFN on HIV-1 viral load (VL). METHODS: Two cohorts of antiretroviral (ART) naïve participants living with HIV (PLWH) with either early (START) or advanced infection (FIRST) were analysed separately. Type 1 IFN genes (n = 17) and receptor subunits (IFNAR1, IFNAR2) were examined for both cumulated type 1 IFN pathway analysis and individual gene analysis. SKAT-O was applied to detect associations between the genotype and HIV-1 study entry viral load (log10 transformed) as a proxy for set point VL; P-values were corrected using Bonferroni (P < 0.0025). RESULTS: The analyses among those with early infection included 2429 individuals from five continents. The median study entry HIV VL was 14,623 (IQR 3460-45100) copies/mL. Across 673 SNPs within 19 type 1 IFN genes, no significant association with study entry VL was detected. Conversely, examining individual genes in START showed a borderline significant association between IFNW1, and study entry VL (P = 0.0025). This significance remained after separate adjustments for age, CD4+ T-cell count, CD4+/CD8+ T-cell ratio and recent infection. When controlling for population structure using linear mixed effects models (LME), in addition to principal components used in the main model, this was no longer significant (p = 0.0244). In subgroup analyses stratified by geographical region, the association between IFNW1 and study entry VL was only observed among African participants, although, the association was not significant when controlling for population structure using LME. Of the 17 SNPs within the IFNW1 region, only rs79876898 (A > G) was associated with study entry VL (p = 0.0020, beta = 0.32; G associated with higher study entry VL than A) in single SNP association analyses. The findings were not reproduced in FIRST participants. CONCLUSION: Across 19 type 1 IFN genes, only IFNW1 was associated with HIV-1 study entry VL in a cohort of ART-naïve individuals in early stages of their infection, however, this was no longer significant in sensitivity analyses that controlled for population structures using LME.

Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

Exercise activates interferon response of the liver via Gpld1 to enhance antiviral innate immunity.

Healthy behavioral patterns could modulate organ functions to enhance the body's immunity. However, how exercise regulates antiviral innate immunity remains elusive. Here, we found that exercise promotes type I interferon (IFN-I) production in the liver and enhances IFN-I immune activity of the body. Despite the possibility that many exercise-induced factors could affect IFN-I production, we identified Gpld1 as a crucial molecule, and the liver as the major organ to promote IFN-I production after exercise. Exercise largely loses the efficiency to induce IFN-I in Gpld1-/- mice. Further studies demonstrated that exercise-produced 3-hydroxybutanoic acid (3-HB) critically induces Gpld1 expression in the liver. Gpld1 blocks the PP2A-IRF3 interaction, thus enhancing IRF3 activation and IFN-I production, and eventually improving the body's antiviral ability. This study reveals that exercise improves antiviral innate immunity by linking the liver metabolism to systemic IFN-I activity and uncovers an unknown function of liver cells in innate immunity.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Profiling of Early Immune Responses to Vaccination Using THP-1-Derived Dendritic Cells.

The COVID-19 pandemic has made assessing vaccine efficacy more challenging. Besides neutralizing antibody assays, systems vaccinology studies use omics technology to reveal immune response mechanisms and identify gene signatures in human peripheral blood mononuclear cells (PBMCs). However, due to their low proportion in PBMCs, profiling the immune response signatures of dendritic cells (DCs) is difficult. Here, we develop a predictive model for evaluating early immune responses in dendritic cells. We establish a THP-1-derived dendritic cell (TDDC) model and stimulate their maturation in vitro with an optimal dose of attenuated yellow fever 17D (YF-17D). Transcriptomic analysis reveals that type I interferon (IFN-I)-induced immunity plays a key role in dendritic cells. IFN-I regulatory biomarkers (IRF7, SIGLEC1) and IFN-I-inducible biomarkers (IFI27, IFI44, IFIT1, IFIT3, ISG15, MX1, OAS2, OAS3) are identified and validated in vitro and in vivo. Furthermore, we apply this TDDC approach to various types of vaccines, providing novel insights into their early immune response signatures and their heterogeneity in vaccine recipients. Our findings suggest that a standardizable TDDC model is a promising predictive approach to assessing early immunity in DCs. Further research into vaccine efficacy assessment approaches on various types of immune cells could lead to a systemic regimen for vaccine development in the future.

Uncoupling interferons and the interferon signature explains clinical and transcriptional subsets in SLE.

Systemic lupus erythematosus (SLE) displays a hallmark interferon (IFN) signature. Yet, clinical trials targeting type I IFN (IFN-I) have shown variable efficacy, and blocking IFN-II failed to treat SLE. Here, we show that IFN type levels in SLE vary significantly across clinical and transcriptional endotypes. Whereas skin involvement correlated with IFN-I alone, systemic features like nephritis associated with co-elevation of IFN-I, IFN-II, and IFN-III, indicating additive IFN effects in severe SLE. Notably, while high IFN-II/-III levels without IFN-I had a limited effect on disease activity, IFN-II was linked to IFN-I-independent transcriptional profiles (e.g., OXPHOS and CD8+GZMH+ cells), and IFN-III enhanced IFN-induced gene expression when co-elevated with IFN-I. Moreover, dysregulated IFNs do not explain the IFN signature in 64% of patients or clinical manifestations including cytopenia, serositis, and anti-phospholipid syndrome, implying IFN-independent endotypes in SLE. This study sheds light on mechanisms underlying SLE heterogeneity and the variable response to IFN-targeted therapies in clinical trials.

Variable Syndromic Immunodeficiency in Patients with Biallelic PRIM1 Mutations.

Mutations in genes of the DNA polymerase complex have been linked to impaired immunological function next to distinct syndromic features. Biallelic mutations in PRIM1 are associated with a primordial dwarfism syndrome with variable hypogammaglobulinemia. The disease is mostly lethal in infancy due to pulmonary infections as well as hepatic cirrhosis. We studied 3 novel patients with PRIM1-deficiency with a focus on immunological consequences. All three shared dysmorphic features including a prominent forehead, triangular face and bilateral cryptorchidism. P1 carried the novel homozygous PRIM1 splice variant c.103+2T>G, allowing residual protein expression and associated with a mild clinical phenotype. P2 and P3 carried the known homozygous variant c.638+36C>G and died in infancy. Paradoxically, B cell lymphopenia was most pronounced in P1. No other significant lymphocyte abnormalities were detected. Interestingly, all 3 patients showed variable, but intermittently excessive Type I interferon signatures. In summary, the B-cell deficiency in PRIM1-deficiency is markedly variable and the severity of syndromic manifestations is not predictive of the immunological phenotype. We highlight a potential contribution of pathological type I interferon activation to disease pathogenesis which warrants further investigations.