Innate Immune Signaling Pathways Research Articles

Development of Self-Adjuvants in mRNA Vaccine and Its Application in Disease Prevention and Treatment.

Adjuvants augment the immunogenicity of vaccines when co-administered with messenger RNA (mRNA) antigens. In recent years, nanotechnology and nanoscience have seen significant growth, resulting in the discovery of synthetic small molecule compounds, natural extracts, and nanomaterials with self-adjuvant properties for nano delivery. The materials exhibit robust immune activity and efficiently activate various innate immune signaling pathways. Moreover, they possess a comparatively simple chemical composition in contrast to conventional adjuvants. This significantly streamlines the manufacturing process of vaccine formulations. Therefore, these self-adjuvant materials theoretically improve the reproducibility of adjuvant production and quality control. Herein, this review summarizes the current research and development progress of mRNA adjuvants, with a specific focus on various types of mRNA adjuvants, notably self-adjuvant nanomaterials. It discusses the current research status on a range of diseases and investigates the potential development of mRNA vaccine adjuvants.

Autophagy activation alleviates the LPS-induced inflammatory response in endometrial epithelial cells in dairy cows.

Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.

Tubercidin inhibits PRRSV replication via RIG-I/NF-κB pathways and interrupting viral nsp2 synthesis.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.

Transcriptional profiling specifies the pathogen-specific human host response to infectious keratitis.

Corneal infections are a leading cause of visual impairment and blindness worldwide. Here we applied high-resolution transcriptomic profiling to assess the general and pathogen-specific molecular and cellular mechanisms during human corneal infection. Clinical diagnoses of herpes simplex virus (HSV) (n=5) and bacterial/fungal (n=5) keratitis were confirmed by histology. Healthy corneas (n=7) and keratoconus (n=4) samples served as controls. Formalin-fixed, paraffin-embedded (FFPE) human corneal specimens were analyzed using the 3' RNA sequencing method Massive Analysis of cDNA Ends (MACE RNA-seq). The cellular host response was investigated using comprehensive bioinformatic deconvolution (xCell and CYBERSORTx) analyses and by integration with published single cell RNA-seq data of the human cornea. Our analysis identified 216 and 561 genes, that were specifically overexpressed in viral or bacterial/fungal keratitis, respectively, and allowed to distinguish the two etiologies. The virus-specific host response was driven by adaptive immunity and associated molecular signaling pathways, whereas the bacterial/fungal-specific host response mainly involved innate immunity signaling pathways and cell types. We identified several genes and pathways involved in the host response to infectious keratitis, including CXCL9, CXCR3, and MMP9 for viral, and S100A8/A9, MMP9, and the IL17 pathway for bacterial/fungal keratitis. High-resolution molecular profiling provides new insights into the human corneal host response to viral and bacterial/fungal infection. Pathogen-specific molecular profiles may provide the foundation for novel diagnostic biomarker and therapeutic approaches that target inflammation-induced damage to corneal host cells with the goal to improve the outcome of infectious keratitis.

In vivo vitamin D target genes interconnect key signaling pathways of innate immunity.

The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), its nuclear receptor VDR (vitamin D receptor) and hundreds of their target genes are not only key regulators of calcium homeostasis, but also important modulators of the immune system. Innate immune cells like monocytes use VDR for efficient differentiation and are very responsive to vitamin D. So far, most information on the gene regulatory function of vitamin D and its physiological impact had been obtained from in vitro studies using supraphysiological doses of 1,25(OH)2D3. Therefore, medical experiments like the study VitDHiD (NCT03537027), where 25 healthy individuals were supplemented once with a vitamin D3 bolus (80,000 IU), provide important insight into the response to vitamin D under in vivo conditions. In this study, we inspected 452 in vivo vitamin D target genes from peripheral blood mononuclear cells (PBMCs) detected in VitDHiD and found 61 of them involved in eight major KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of innate immunity. Under in vivo conditions in healthy individuals vitamin D either silences five pathways of innate immunity, stabilizes two and increases one, so that acute inflammation is suppressed and the release of cytokines is kept under control. A ranking of the 61 target genes by inducibility, basal expression and multiple involvements in the pathways highlighted the genes NFKBIA (NFκB inhibitor alpha), NFKBIZ, FOSL2 (FOS like 2, AP1 transcription factor subunit), JDP2 (Jun dimerization protein 2), PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1), CLEC7A (C-type lectin domain containing 7A), DUSP6 (dual specificity phosphatase 6), NCF2 (neutrophil cytosolic factor 2), PLCB1 (phospholipase C beta 1), PLCG2 and TNFAIP3 (TNF alpha induced protein 3). In conclusion, vitamin D's in vivo effect on innate immunity in healthy adults is mediated by the interconnection of the pathways of neutrophil extracellular trap formation, Toll-like receptor, chemokine and phagosome signaling, NOD-like receptor, C-type lectin receptor, apoptosis and interleukin 17 through a limited set of proteins encoded by key target genes.

WEE1 Inhibitors Mediate Antitumor Effects on Endometrial Cancer through Activation of Innate Immune Responses.

Introduction: Recurrence signifies the primary mortality factor in patients suffering from endometrial cancer, with few efficacious treatments currently available for recurrent cases. This research investigates the anti-tumoral capacities of WEE1 inhibitors within the context of endometrial cancer, aiming to establish a novel therapeutic avenue for high recurrence-risk patients. Materials and methods: We evaluated WEE1 expression in endometrial cancer patients utilizing immunohistochemistry on paraffin-embedded tissue sections. The cytotoxic potential of WEE1 inhibitors on endometrial cancer cells was assessed by CCK8 assay. Assays to gauge the influence of WEE1 inhibitors on cell proliferation and migration included clonal proliferation, wound healing, and transwell assays. We determined the impacts on apoptosis and cell cycle stages by flow cytometry. Employing qRT-PCR and western blotting, we investigated the mechanistic pathways underlying the anti-tumoral activity of WEE1 inhibitors. In vivo evaluations were executed to ascertain the inhibitory effect of WEE1 inhibitors on tumor growth in mice. Results: WEE1 exhibited high-level expression in endometrial cancer tissues, particularly pronounced in recurrent compared with non-recurrent patients. WEE1 inhibitors effectively eliminated endometrial cancer cells while inhibiting their proliferation and migration. Flow cytometric analyses revealed a significant promotion of apoptosis and an increase in G2/M phase cell proportion upon WEE1 inhibitor treatment. qRT-PCR and western blotting elucidated that WEE1 inhibitors activated the innate immune signaling pathway in endometrial cancer cells. Furthermore, in vivo assessments demonstrated substantial tumor growth suppression due to WEE1 inhibitors. Conclusions: WEE1 inhibitors initiated an innate immune response in endometrial cancer, exhibiting considerable anti-tumoral effects, which was promising for postoperative treatment of endometrial cancer, especially recurrent endometrial cancer patients.

Cytoplasmic DNA and AIM2 inflammasome in RA: where they come from and where they go?

Rheumatoid arthritis is a chronic autoimmune disease of undetermined etiology characterized by symmetric synovitis with predominantly destructive and multiple joint inflammation. Cytoplasmic DNA sensors that recognize protein molecules that are not themselves or abnormal dsDNA fragments play an integral role in the generation and perpetuation of autoimmune diseases by activating different signaling pathways and triggering innate immune signaling pathways and host defenses. Among them, melanoma deficiency factor 2 (AIM2) recognizes damaged DNA and double-stranded DNA and binds to them to further assemble inflammasome, initiating the innate immune response and participating in the pathophysiological process of rheumatoid arthritis. In this article, we review the research progress on the source of cytoplasmic DNA, the mechanism of assembly and activation of AIM2 inflammasome, and the related roles of other cytoplasmic DNA sensors in rheumatoid arthritis.

RanBP2/Nup358 Mediates Sumoylation of STAT1 and Antagonizes Interferon-α-Mediated Antiviral Innate Immunity.

Type I interferon (IFN-I)-induced signaling plays a critical role in host antiviral innate immune responses. Despite this, the mechanisms that regulate this signaling pathway have yet to be fully elucidated. The nucleoporin Ran Binding Protein 2 (RanBP2) (also known as Nucleoporin 358 KDa, Nup358) has been implicated in a number of cellular processes, including host innate immune signaling pathways, and is known to influence viral infection. In this study, we documented that RanBP2 mediates the sumoylation of signal transducers and activators of transcription 1 (STAT1) and inhibits IFN-α-induced signaling. Specifically, we found that RanBP2-mediated sumoylation inhibits the interaction of STAT1 and Janus kinase 1 (JAK1), as well as the phosphorylation and nuclear accumulation of STAT1 after IFN-α stimulation, thereby antagonizing the IFN-α-mediated antiviral innate immune signaling pathway and promoting viral infection. Our findings not only provide insights into a novel function of RanBP2 in antiviral innate immunity but may also contribute to the development of new antiviral therapeutic strategies.

Ubiquitin signalling in Drosophila innate immune responses.

Cells respond to invading pathogens and danger signals from the environment by adapting gene expression to meet the need for protective effector molecules. While this innate immune response is required for the cell and the organism to recover, excess immune activation may lead to loss of homeostasis, thereby promoting chronic inflammation and cancer progression. The molecular basis of innate immune defence is comprised of factors promoting survival and proliferation, such as cytokines, antimicrobial peptides and anti-apoptotic proteins. As the molecular mechanisms regulating innate immune responses are conserved through evolution, the fruit fly Drosophila melanogaster serves as a convenient, affordable and ethical model organism to enhance understanding of immune signalling. Fly immunity against bacterial infection is built up by both cellular and humoral responses, where the latter is regulated by the Imd and Toll pathways activating NF-κB transcription factors Relish, Dorsal and Dif, as well as JNK activation and JAK/STAT signalling. As in mammals, the Drosophila innate immune signalling pathways are characterised by ubiquitination of signalling molecules followed by ubiquitin receptors binding to the ubiquitin chains, as well as by rapid changes in protein levels by ubiquitin-mediated targeted proteasomal and lysosomal degradation. In this review, we summarise the molecular signalling pathways regulating immune responses to pathogen infection in Drosophila, with a focus on ubiquitin-dependent control of innate immunity and inflammatory signalling.

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA.

The Picornaviridae family comprises a large group of non-enveloped viruses with enormous impact on human and animal health. The picornaviral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteases. The picornaviral 3C proteases share similar three-dimensional structures and play a significant role in the viral life cycle and virus-host interactions. Picornaviral 3C proteins also have conserved RNA-binding activities that contribute to the assembly of the viral RNA replication complex. The 3C protease is important for regulating the host cell response through the cleavage of critical host cell proteins, acting to selectively 'hijack' host factors involved in gene expression, promoting picornavirus replication, and inactivating key factors in innate immunity signaling pathways. The protease and RNA-binding activities of 3C are involved in viral polyprotein processing and the initiation of viral RNA synthesis. Most importantly, 3C modifies critical molecules in host organelles and maintains virus infection by subtly subverting host cell death through the blocking of transcription, translation, and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Here, we discuss the molecular mechanisms through which 3C mediates physiological processes involved in promoting virus infection, replication, and release.

The emerging roles of protein arginine methyltransferases in antiviral innate immune signaling pathways.

The Protein Arginine Methyltransferases (PRMTs) family is involved in various biological processes, including gene transcription, pre-mRNA splicing, mRNA translation, and protein stability. Recently, mounting evidence has shown that PRMTs also play critical roles in regulating the host antiviral immune response, either in an enzymatic activity dependent or independent manner. This review aims to provide an overview of the recent findings regarding the function and regulatory mechanisms of PRMTs in the antiviral response. These findings have the potential to aid in the discovery and design of novel therapeutic strategies for viral infections.

Mitochondrial nucleic acids in innate immunity and beyond.

Mitochondria participate in a wide range of cellular processes. One essential function of mitochondria is to be a platform for antiviral signaling proteins during the innate immune response to viral infection. Recently, studies have revealed that mitochondrion-derived DNAs and RNAs are recognized as non-self molecules and act as immunogenic ligands. More importantly, the cytosolic release of these mitochondrial nucleic acids (mt-NAs) is closely associated with the pathogenesis of human diseases accompanying aberrant immune activation. The release of mitochondrial DNAs (mtDNAs) via BAX/BAK activation and/or VDAC1 oligomerization activates the innate immune response and inflammasome assembly. In addition, mitochondrial double-stranded RNAs (mt-dsRNAs) are sensed by pattern recognition receptors in the cytosol to induce type I interferon expression and initiate apoptotic programs. Notably, these cytosolic mt-NAs also mediate adipocyte differentiation and contribute to mitogenesis and mitochondrial thermogenesis. In this review, we summarize recent studies of innate immune signaling pathways regulated by mt-NAs, human diseases associated with mt-NAs, and the emerging physiological roles of mt-NAs.

Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus.

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-β, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host's innate immune response.

Cell-type-specific need of Ddx3 and PACT for interferon induction by RNA viruses.

Interferon-stimulated genes (ISGs) are induced in response to interferon expression due to viral infections. Role of these ISGs can be variable in different cells or organs. Our study highlights such cell-specific role of an ISG, Ddx3, which regulates the translation of mRNAs essential for interferon induction (PACT) and interferon signaling (STAT1) in a cell-specific manner. Our study also highlights the role of PACT in RNA virus-induced RLR signaling. Our study depicts how Ddx3 regulates innate immune signaling pathways in an indirect manner. Such cell-specific behavior of ISGs helps us to better understand viral pathogenesis and highlights the complexities of viral tropism and innate immune responses.

DLG1 promotes the antiviral innate immune response by inhibiting p62-mediated autophagic degradation of IKKε.

The type-I interferon (IFN-I) signaling pathway is the first line of antiviral innate immunity. It must be precisely regulated against virus-induced damage. The tightly regulated mechanisms of action of host genes in the antiviral innate immune signaling pathway are still worth studying. Here, we report a novel role of DLG1 in positively regulating the IκB kinase epsilon (IKKε)-mediated IFN-I signaling response against negative-stranded RNA virus replication, whereas the RNA virus inhibits the expression of DLG1 for immune escape. Importantly, the E3 ligase March2 interacts with and promotes K27-linked polyubiquitination of IKKε, and p62 is a cargo receptor that recognizes ubiquitinated IKKε for eventual autophagic degradation. Together, the current findings elucidate the role of DLG1 in the antiviral IFN-I signaling pathway and viral infection repression.

Chicken IFI6 inhibits avian reovirus replication and affects related innate immune signaling pathways.

Interferon-alpha inducible protein 6 (IFI6) is an important interferon-stimulated gene. To date, research on IFI6 has mainly focused on human malignant tumors, virus-related diseases and autoimmune diseases. Previous studies have shown that IFI6 plays an important role in antiviral, antiapoptotic and tumor-promoting cellular functions, but few studies have focused on the structure or function of avian IFI6. Avian reovirus (ARV) is an important virus that can exert immunosuppressive effects on poultry. Preliminary studies have shown that IFI6 expression is upregulated in various tissues and organs of specific-pathogen-free chickens infected with ARV, suggesting that IFI6 plays an important role in ARV infection. To analyze the function of avian IFI6, particularly in ARV infection, the chicken IFI6 gene was cloned, a bioinformatics analysis was conducted, and the roles of IFI6 in ARV replication and the innate immune response were investigated after the overexpression or knockdown of IFI6 in vitro. The results indicated that the molecular weight of the chicken IFI6 protein was approximately 11 kDa and that its structure was similar to that of the human IFI27L1 protein. A phylogenetic tree analysis of the IFI6 amino acid sequence revealed that the evolution of mammals and birds was clearly divided into two branches. The evolutionary history and homology of chickens are similar to those of other birds. Avian IFI6 localized to the cytoplasm and was abundantly expressed in the chicken lung, intestine, pancreas, liver, spleen, glandular stomach, thymus, bursa of Fabricius and trachea. Further studies demonstrated that IFI6 overexpression in DF-1 cells inhibited ARV replication and that the inhibition of IFI6 expression promoted ARV replication. After ARV infection, IFI6 modulated the expression of various innate immunity-related factors. Notably, the expression patterns of MAVS and IFI6 were similar, and the expression patterns of IRF1 and IFN-β were opposite to those of IFI6. The results of this study further advance the research on avian IFI6 and provide a theoretical basis for further research on the role of IFI6 in avian virus infection and innate immunity.

Kme-0584, a Highly Potent IRAK1/IRAK4/panFLT3 Inhibitor, Is a Promising Clinical Candidate for Hypomethylating Agent Plus Venetoclax Resistant AML/MDS Patients

Leukemic cells exhibit dysregulation of innate immune signaling pathways upon diagnosis, and these pathways become further activated in drug resistance. The Interleukin-1 receptor associated kinase 1 and 4 (IRAK1/IRAK4) kinase complex is part of a critical signaling node that becomes activated in these dysregulated pathways (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK4 inhibitors are currently under investigation for the treatment of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and have shown encouraging, though modest responses in clinical studies. We have recently demonstrated that limited responses to IRAK4 inhibitors in the leukemic setting can be explained by a compensated upregulation and activation of IRAK1, and the need to inhibit both IRAK1 and IRAK4 to achieve maximal therapeutic efficacy (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). KME-0584 is a highly potent IRAK1/IRAK4/panFLT3 inhibitor that exhibits superior potency and therapeutic efficacy vs. IRAK4 inhibitor compounds in the FLT3 WT as well as the FLT3 mutant setting. KME-0584 exhibits >100-fold selectivity vs. 89% of the Kinome as measured in the Reaction Biology 374 kinase panel, with an IC50 of 23, <1.29, and <0.5nM at IRAK1, IRAK4, and FLT3 respectively. Higher kinase selectivity is seen in cell-based assays vs. biochemical assays, where high potency kinase antagonist activity is also observed against PHKg1, PDGFRβ, RET, CLK1, and CLK4. While IRAK4-selective compounds fully antagonize NF-κB signaling through the TLR pathway, KME-0584 completely inhibits NF-κB signaling through both the TLR and IL1-receptor pathways, indicating complete inhibition of multiple receptor pathways converging on NF-κB requires both IRAK1 and IRAK4 antagonism. In primary patient cell lines from FLT3 wildtype (WT) patients, KME-0584 inhibits leukemia stem cell progenitor function as measured by the colony formation assay in methylcellulose with higher potency than IRAK4 inhibitor compounds that lack IRAK1 activity such as CA-4948 (Emavusertib). This activity is independent of patient mutational status. Importantly, high potency in primary patient cell lines is observed independent of the presence of U2AF1 or SF3B1 spliceosome mutations known to drive expression of the activated IRAK4 Long isoform of IRAK4 (Fig 1), implying that the IRAK1/4/panFLT3 inhibitor KME-0584 will have activity across a broader patient population than an IRAK4 inhibitor. We have previously described an IRAK1/4 gene signature that is highly expressed in patients with myelomonocytic subtypes (M4 FAB) of adult and pediatric AML and with an antecedent MDS (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). The IRAK1/4 gene signature is reversed after treatment of THP1 cells for 24 hours with KME-0584. Given that monocytic-like subtypes of AML are resistant to Venetoclax plus Azacitidine (VEN/AZA) (S Pei et. al. Cancer Discovery, 2020), these data suggest that KME-0584 has the potential to be effective across a broader range of relapsed/refractory AML/MDS patients than previously examined IRAK4 inhibitors. KME-0584 exhibits superior potency and efficacy to gilteritinib in the FLT3-ITD (D835Y) xenograft model after QD oral dosing, with sufficient PK and oral bioavailability across multiple species to support QD or BID dosing in the clinic. KME-0584 does not inhibit any of the major or minor cytochrome P450 enzymes at anticipated clinical concentrations and early indication from ongoing GLP toxicology studies suggest that it could be safely administered in humans. A clinical study of KME-0584 in relapsed/refractory AML and HR-MDS is currently planned to start in 1H 2024.

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

Toxoplasma gondii is an important intracellular protozoan, which needs to exploit host nutrition for successful parasitism. We previously reported that Casitas B-lineage lymphoma-b (Cbl-b) was screened as a host dependency factor of T. gondii by using lentiviral CRISPR-Cas9-single guide RNA (sgRNA) libraries. Furthermore, the detailed mechanism of Cbl-b being required by T. gondii infection was explored in this study. The proliferation of T. gondii was found to be significantly inhibited in Cbl-b knockdown cell lines, and the Cbl-b expression level increased with prolonged T. gondii infection, while the MyD88 level was significantly decreased in the T. gondii infection group. Toll-like receptor (TLR)/MyD88 is a conserved innate cellular immune signaling pathway against pathogens infection. Cbl-b was found to interact with MyD88 and mediate MyD88 ubiquitination in the fluorescence resonance energy transfer and co-immunoprecipitation experiments. A Cbl-b knockout (KO) C57BL/6J lineage was then constructed and, together with the wild-type (WT) mice, was infected with the same amount of T. gondii tachyzoites of ME49 strain. At 13 days post infection, all the mice in the WT group died, while 80% of the mice in the Cbl-b KO group still survived, even to the end of the experiment. The parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that of the WT mice (P < 0.05). In Cbl-b KO infected mice, the percentage of B cells was higher, whereas that of macrophages was lower, and the interferon-γ and interleukin-6 levels in the serum were higher than that in the WT infected mice; the difference was significant (P < 0.05). Furthermore, when host cells were infected by T. gondii, host's Cbl-b was found to interact with MyD88 to ubiquitinate MyD88 for ribosome-dependent degradation. Therefore, the host's innate immunity against T. gondii through the TLR/MyD88 pathway was negatively regulated by Cbl-b.IMPORTANCEThis is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

Mosquito-borne flaviviruses and type I interferon: catch me if you can!

Mosquito-borne flaviviruses include many viruses that are important human pathogens, including Yellow fever virus, Dengue virus, Zika virus and West Nile virus. While these viruses have long been confined to tropical regions, they now pose a global public health concern, as the geographical distribution of their mosquito vectors has dramatically expanded. The constant threat of flavivirus emergence and re-emergence underlines the need for a better understanding of the relationships between these viruses and their hosts. In particular, unraveling how these viruses manage to bypass antiviral immune mechanisms could enable the design of countermeasures to limit their impact on human health. The body's first line of defense against viral infections is provided by the interferon (IFN) response. This antiviral defense mechanism takes place in two waves, namely the induction of type I IFNs triggered by viral infection, followed by the IFN signaling pathway, which leads to the synthesis of interferon-stimulated genes (ISGs), whose products inhibit viral replication. In order to spread throughout the body, viruses must race against time to replicate before this IFN-induced antiviral state hinders their dissemination. In this review, we summarize our current knowledge on the multiple strategies developed by mosquito-borne flaviviruses to interfere with innate immune detection and signaling pathways, in order to delay, if not prevent, the establishment of an antiviral response.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.