Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Opposing Functions of Interferon Coordinate Adaptive and Innate Immune Responses to Cancer Immune Checkpoint Blockade.

Interferon-gamma (IFNG) augments immune function yet promotes T cell exhaustion through PDL1. How these opposing effects are integrated to impact immune checkpoint blockade (ICB) is unclear. We show that while inhibiting tumor IFNG signaling decreases interferon-stimulated genes (ISGs) in cancer cells, it increases ISGs in immune cells by enhancing IFNG produced by exhausted T cells (TEX). In tumors with favorable antigenicity, these TEX mediate rejection. In tumors with neoantigen or MHC-I loss, TEX instead utilize IFNG to drive maturation of innate immune cells, including a PD1+TRAIL+ ILC1 population. By disabling an inhibitory circuit impacting PD1 and TRAIL, blocking tumor IFNG signaling promotes innate immune killing. Thus, interferon signaling in cancer cells and immune cells oppose each other to establish a regulatory relationship that limits both adaptive and innate immune killing. In melanoma and lung cancer patients, perturbation of this relationship is associated with ICB response independent of tumor mutational burden.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

An Antiviral Branch of the IL-1 Signaling Pathway Restricts Immune-Evasive Virus Replication.

Virulent pathogens often cause the release of host-derived damage-associated molecular patterns (DAMPs) from infected cells. During encounters with immune-evasive viruses that block inflammatory gene expression, preformed DAMPs provide backup inflammatory signals that ensure protective immunity. Whether DAMPs exhibit additional backup defense activities is unknown. Herein, we report that viral infection of barrier epithelia (keratinocytes) elicits the release of preformed interleukin-1 (IL-1) family cytokines, including the DAMP IL-1α. Mechanistic studies revealed that IL-1 acts on skin fibroblasts to induce an interferon (IFN)-like state that restricts viral replication. We identified a branch in the IL-1 signaling pathway that induces IFN-stimulated gene expression in infected cells and found that IL-1 signaling is necessary to restrict viral replication in human skin explants. These activities are most important to control immune-evasive virus replication in fibroblasts and other barrier cell types. These findings highlight IL-1 as an important backup antiviral system to ensure barrier defense.

The immunobiology of corneal HSV-1 infection and herpetic stromal keratitis.

SUMMARYHuman alphaherpesvirus 1 (HSV-1) is a highly successful neurotropic pathogen that primarily infects the epithelial cells lining the orofacial mucosa. After primary lytic replication in the oral, ocular, and nasal mucosal epithelial cells, HSV-1 establishes life-long latency in neurons within the trigeminal ganglion. Patients with compromised immune systems experience frequent reactivation of HSV-1 from latency, leading to virus entry in the sensory neurons, followed by anterograde transport and lytic replication at the innervated mucosal epithelial surface. Although recurrent infection of the corneal mucosal surface is rare, it can result in a chronic immuno-inflammatory condition called herpetic stromal keratitis (HSK). HSK leads to gradual vision loss and can cause permanent blindness in severe untreated cases. Currently, there is no cure or successful vaccine to prevent latent or recurrent HSV-1 infections, posing a significant clinical challenge to managing HSK and preventing vision loss. The conventional clinical management of HSK primarily relies on anti-virals to suppress HSV-1 replication, anti-inflammatory drugs (such as corticosteroids) to provide symptomatic relief from pain and inflammation, and surgical interventions in more severe cases to replace damaged cornea. However, each clinical treatment strategy has limitations, such as local and systemic drug toxicities and the emergence of anti-viral-resistant HSV-1 strains. In this review, we summarize the factors and immune cells involved in HSK pathogenesis and highlight alternate therapeutic strategies for successful clinical management of HSK. We also discuss the therapeutic potential of immunoregulatory cytokines and immunometabolism modulators as promising HSK therapies against emerging anti-viral-resistant HSV-1 strains.

An essential role for Cmtr2 in mammalian embryonic development.

CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function.

Age-related transcript changes in type I interferon signaling in children and adolescents with long COVID.

SARS-CoV-2 typically causes mild symptoms in children, but evidence suggests that persistent immunopathological changes may lead to long COVID (LC). To explore the interplay between LC and innate immunity, we assessed the type I interferon (IFN-I) response in children and adolescents with LC symptoms (LC; n = 28). This was compared with age-matched SARS-CoV-2 recovered participants without LC symptoms (MC; n = 28) and healthy controls (HC; n = 18). We measured the mRNA expression of IFN-I (IFN-α/ß/ε/ω), IFN-I receptor (IFNAR1/2), and ISGs (ISG15, ISG56, MxA, IFI27, BST2, LY6E, OAS1, OAS2, OAS3, and MDA5) in PBMCs collected 3-6 months after COVID-19. LC adolescents (12-17 years) had higher transcript levels of IFN-ß, IFN-ε, and IFN-ω than HC, whereas LC children (6-11 years) had lower levels than HC. In adolescents, increased levels of IFN-α, IFN-ß, and IFN-ω mRNAs were found in the LC group compared with MC, while lower levels were observed in LC children than MC. Adolescents with neurological symptoms had higher IFN-α/ß mRNA levels than MC. LC and MC participants showed decreased expression of ISGs and IFNAR1, but increased expression of IFNAR2, than HC. Our results show age-related changes in the expression of transcripts involved in the IFN-I signaling pathway in children and adolescents with LC.

African swine fever virus pH240R enhances viral replication via inhibition of the type I IFN signaling pathway.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by ASF virus (ASFV) infection. At present, there are still no safe and effective drugs and vaccines to prevent ASF. Mining the important proteins encoded by ASFV that affect the virulence and replication of ASFV is the key to developing effective vaccines and drugs. In this study, ASFV pH240R, a capsid protein of ASFV, was found to inhibit the type I interferon (IFN) signaling pathway. Mechanistically, pH240R interacted with IFNAR1 and IFNAR2 to disrupt the interaction of IFNAR1-TYK2 and IFNAR2-JAK1. Additionally, pH240R inhibited the phosphorylation of IFNAR1, TYK2, and JAK1 induced by IFN-α, resulting in the suppression of the nuclear import of STAT1 and STAT2 and the expression of IFN-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induced more ISGs in porcine alveolar macrophages compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs expression. Taken together, our results clarify that pH240R enhances ASFV replication by inhibiting the JAK-STAT signaling pathway, which highlights the possibility of pH240R as a potential drug target.IMPORTANCEThe innate immune response is the host's first line of defense against pathogen infection, which has been reported to affect the replication and virulence of African swine fever virus (ASFV) isolates. Identification of ASFV-encoded proteins that affect the virulence and replication of ASFV is the key step in developing more effective vaccines and drugs. In this study, we found that pH240R interacted with IFNAR1 and IFNAR2 by disrupting the interaction of IFNAR1-TYK2 and IFNAR2-JAK1, resulting in the suppression of the expression of interferon (IFN)-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induces more ISGs' expression compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs' expression. Taken together, our findings showed that pH240R enhances ASFV replication by inhibiting the IFN-JAK-STAT axis, which highlights the possibility of pH240R as a potential drug target.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Latent gammaherpesvirus infection enhances type I IFN response and reduces virus spread in an influenza A virus co-infection model.

Infections with persistent or latent viruses alter host immune homeostasis and have potential to affect the outcome of concomitant acute viral infections such as influenza A virus (IAV). Gammaherpesviruses establish life-long infections and require an on-going immune response to control reactivation. We have used a murine model of co-infection to investigate the response to IAV infection in mice latently infected with the gammaherpesvirus MHV-68. Over the course of infection, latently infected BALB/c mice showed less weight loss, clinical signs, pulmonary cellular infiltration and expression of inflammatory mediators than naïve mice infected with IAV and had significantly more activated CD8+ T cells in the lungs. Four days after IAV infection, virus spread in the lungs of latently infected animals was significantly lower than in naïve animals. By 7 days after IAV infection latently infected lungs express elevated levels of cytokines and chemokines indicating they are primed to respond to the secondary infection. Investigation at an early time point showed that 24 h after IAV infection co-infected animals had higher expression of IFNß and Ddx58 (RIG-I) and a range of ISGs than mice infected with IAV alone suggesting that the type I IFN response plays a role in the protective effect. This effect was mouse strain dependent and did not occur in 129/Sv/Ev mice. These results offer insight into innate immune mechanisms that could be utilized to protect against IAV infection and highlight on-going and persistent viral infections as a significant factor impacting the severity of acute respiratory infections.

Guanylate-binding protein 1 inhibits Hantaan virus infection by restricting virus entry.

Hantaan virus (HTNV) infection can cause hemorrhagic fever with renal syndrome (HFRS) in humans, and currently, there are no long-standing protective vaccines or specific antivirals available. Guanylate-binding protein 1 (GBP1) is an interferon-stimulated gene that defends against various pathogen infections. However, the function of GBP1 in HTNV infection remains unknown. Here, we describe how GBP1 prevents HTNV infection by obstructing virus entry. We found that HTNV infection induced GBP1 expression and that overexpression of GBP1 inhibited HTNV infection, while knockout of GBP1 had the opposite effect. Interestingly, GBP1 did not affect interferon (IFN) signaling during HTNV infection. Instead, GBP1 prevented HTNV from entering cells through clathrin-mediated endocytosis (CME). We also discovered that GBP1 specifically interacted with actin but not dynamin 2 (DNM2) and made it difficult for DNM2 to be recruited by actin, which may account for the suppression of CME during HTNV infection. These findings establish an antiviral role for GBP1 in inhibiting HTNV infection and help us better understand how GBP1 regulates HTNV entry and could potentially aid in developing treatments for this virus.

Evaluation of long non-coding RNAs EGOT, NRAV, NRIR and mRNAs ISG15 and IFITM3 expressions in COVID-19 patients.

Individuals with Coronavirus Disease 2019 (COVID-19) may show no symptoms to moderate or severe complications. This variation may be due to differences in the strength of the immune response, including a delayed interferon (IFN) response in asymptomatic patients and higher IFN levels in severe patients. Some long non-coding RNAs (lncRNAs), as regulators of the IFN pathway, may contribute to the emergence of different COVID-19 symptoms. This study aimed to comparatively investigate the relationship between lncRNAs (eosinophil granule ontogeny transcript (EGOT), negative regulator of antiviral response (NRAV), and negative regulator of interferon response (NRIR)), alongside interferon-stimulated genes (ISGs) like ISG-15 and interferon-induced transmembrane protein 3 (IFITM3) in COVID-19 patients with asymptomatic, moderate, and severe symptoms. Buffy coat samples were collected from 17 asymptomatic, 23 moderate, 22 severe patients, and 44 healthy controls. Quantitative real-time PCR was utilized to determine the expression levels. In a comparison between COVID-19 patients and healthy individuals, higher expression levels of EGOT and NRAV were observed in severe and moderate patients. NRIR expression was increased across all patient groups. Meanwhile, ISG15 expression decreased in all patient groups, and the moderate group showed a significant decrease in IFITM3 expression. Comparing COVID-19 patient groups, EGOT expression was significantly higher in moderate COVID-19 patients compared to asymptomatic patients. NRAV was higher in moderate and severe patients compared to asymptomatic. NRIR levels did not differ significantly between the COVID-19 patient groups. ISG15 was higher in moderate and severe patients compared to asymptomatic. IFITM3 expression was significantly higher in severe patients compared to the moderate group. In severe COVID-19 patients, EGOT expression was positively correlated with NRAV levels. EGOT and NRAV showed a significant positive correlation in asymptomatic patients, and both were positively correlated with IFITM3 expression. This study suggests that EGOT, NRAV, NRIR, ISG15, and IFITM3 may serve as diagnostic biomarkers for COVID-19. The lncRNA NRAV may be a good biomarker in a prognostic panel between asymptomatic and severe patients in combination with other high-sensitivity biomarkers. EGOT, NRAV, and ISG15 could also be considered as specific biomarkers in a prognostic panel comparing asymptomatic and moderate patients with other high-sensitivity biomarkers.

Type II interferons (IFN-γ and IFN-γrel) activate downstream genes through various potential receptor combinations to exert antiviral functions in spotted sea bass (Lateolabrax maculatus).

Type II interferons (IFNs) exert antiviral functions by binding to receptors and activating downstream signaling pathways. However, our understanding of the antiviral functions and the receptor complex model of type II IFNs in teleost fish remains limited. In this study, we determined the functions of type II IFNs (LmIFN-γ and LmIFN-γrel) in Lateolabrax maculatus and assessed their antiviral ability mediated by their combination with different cytokine receptor family B members (LmCRFB6, LmCRFB13, and LmCRFB17). After infection with largemouth bass ulcer syndrome virus (LBUSV), the expression levels of LmIFNs and LmCRFBs increased significantly in vitro and in vivo. Incubation or injection with LmIFNs-His activated the expressions of LmISG15, LmMx, and LmIRF1. LmIFN-γ and LmIFN-γrel both bound to the extracellular domains of the three CRFBs via Pull-down. Furthermore, LmIFN-γ combined with LmCRFB6, LmCRFB6+LmCRFB13, and LmCRFB6+LmCRFB13+LmCRFB17 and LmIFN-γrel combined with all combinations containing LmCRFB17 induced the transcription of downstream genes and reduced the number of LBUSV copies. Therefore, type II IFNs (LmIFN-γ and LmIFN-γrel) contribute to enhanced antiviral immunity in L. maculatus and that ligand-receptor combinations effectively suppress virus replication. These findings provide a reference for future studies of the signal transduction mechanism of type II IFNs in teleost fish.

VRK1 promotes DNA-induced type I interferon production.

BACKGROUND: Type I interferons (IFNs) are an essential class of cytokines with antitumor, antiviral and immunoregulatory effects. However, over-productive the type I IFNs are tightly associated with autoimmune disorders. Thus, the induction of type I interferons is precisely regulated to maintain immune hemostasis. This study aimed to identify a novel regulator of type I interferon signaling. METHODS AND RESULTS: Primary BMDMs, isolated from mice, and human cell lines (HEK293 cells, Hela cells) and murine cell line (MEF cells) were cultured to generate in vitro models. After knockdown VRK1, real-time PCR and dual-luciferase reporter assay were performed to determine the expression level of the type I IFNs and ISGs following HTDNA and Poly (dA:dT) stimulation. Additionally, cells were treated with the VRK1 inhibitor, and the impact of VRK1 inhibition was detected. Upon HTDNA and Poly (dA:dT) stimulation, knockdown of VRK1 attenuated the induction of the type I IFNs and ISGs. Consistently, VRK-IN-1, a potent and selective VRK1 inhibitor, significantly suppressed the induction of the type I IFNs and ISGs in human and murine cell lines. Further, VRK-IN-1 inhibited induction of the type I IFNs in mouse primary BMDMs. Intriguingly, VRK1 potentiated the cGAS-STING- IFN-I axis response at STING level. CONCLUSIONS: Our study reveals a novel function of VRK1 in regulating the production of type I IFNs. VRK-IN-1 might be a potential lead compound for suppressing aberrant type I IFNs in autoimmune disorders.

Viral RNA Is a Hub for Critical Host-Virus Interactions.

RNA is a central molecule in the life cycle of viruses, acting not only as messenger (m)RNA but also as a genome. Given these critical roles, it is not surprising that viral RNA is a hub for host-virus interactions. However, the interactome of viral RNAs remains largely unknown. This chapter discusses the importance of cellular RNA-binding proteins in virus infection and the emergent approaches developed to uncover and characterise them.

Recent advances in early pregnancy loss diagnosis in dairy cows: New approaches.

Early pregnancy loss is a primary cause of low reproductive rates in dairy cows, posing severe economic losses to dairy farming. The accurate diagnosis of dairy cows with early pregnancy loss allows for oestrus synchronization, shortening day open, and increasing the overall conception rate of the herd. Several techniques are available for detecting early pregnancy loss in dairy cows, including rectal ultrasound, circulating blood progesterone, and pregnancy-associated glycoproteins (PAGs). Yet, there is a need to improve on existing techniques and develop novel strategies to identify cows with early pregnancy loss accurately. This manuscript reviews the applications of rectal ultrasound, circulating blood progesterone concentration, and PAGs in the diagnosis of pregnancy loss in dairy cows. The manuscript also discusses the recent progress of new technologies, including colour Doppler ultrasound (CDUS), interferon tau-induced genes (ISGs), and exosomal miRNA in diagnosing pregnancy loss in dairy cows. This study will provide an option for producers to re-breed cows with pregnancy loss, thereby reducing the calving interval and economic costs. Meanwhile, this manuscript might also act as a reference for exploring more economical and precise diagnostic technologies for early pregnancy loss in dairy cows.

Transcriptome Sequencing Reveals Salmonella Flagellin Activation of Interferon-ß-Related Immune Responses in Macrophages.

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-1ß, 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)-ß. 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-ß and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-ß-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.

Hypoxia dampens innate immune signalling at early time points and increases Zika virus RNA levels in iPSC-derived macrophages.

Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under laboratory conditions of 18% oxygen (O2), that do not reflect the oxygen levels in most organs (1-5 % O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) infection. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Hypoxic conditions increased the abundance of ZIKV RNA highlighting the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defences.

Cytosolic DNA sensor activation inhibits HIV infection of macrophages.

Cytosolic recognition of microbial DNA in macrophages results in the activation of the interferon (IFN)-dependent antiviral innate immunity. Here, we examined whether activating DNA sensors in peripheral blood monocyte-derived macrophages (MDMs) can inhibit human immunodeficiency virus (HIV). We observed that the stimulation of MDMs with poly(dA:dT) or poly(dG:dC) (synthetic ligands for the DNA sensors) inhibited HIV infection and replication. MDMs treated with poly(dA:dT) or poly(dG:dC) expressed higher levels of both type I and type III IFNs than untreated cells. Activation of the DNA sensors in MDMs also induced the expression of the multiple intracellular anti-HIV factors, including IFN-stimulated genes (ISGs: ISG15, ISG56, Viperin, OAS2, GBP5, MxB, and Tetherin) and the HIV restriction microRNAs (miR-29c, miR-138, miR-146a, miR-155, miR-198, and miR-223). In addition, the DNA sensor activation of MDM upregulated the expression of the CC chemokines (RANTES, MIP-1α, MIP-1ß), the ligands for HIV entry coreceptor CCR5. These observations indicate that the cytosolic DNA sensors have a protective role in the macrophage intracellular immunity against HIV and that targeting the DNA sensors has therapeutic potential for immune activation-based anti-HIV treatment.