Pattern Recognition Receptor Signaling Research Articles

Species-specific responses during Seoul orthohantavirus infection in human and rat lung microvascular endothelial cells.

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.

Abstract 5799: Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response

Abstract Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathways is associated with tumor growth and progression. EGFR overexpression occurs in various types of solid cancers and correlates with poor outcome. Unfortunately, the clinical benefit from EGFR-targeted therapies is usually short-lived, due to the development of resistance. TargImmune has developed first-in-class Targeted Apoptotic Immunomodulators (TAIMs), which harness the body’s anti-viral responses to treat solid tumors. Our novel nanoparticle platform technology, Ta:RNA , selectively delivers synthetic dsRNA, polyinosinic:polycytidylic acid (polyIC) to tumor cells that overexpress a target receptor. PolyIC and other pattern recognition receptor (PRR) agonists have been used as adjuvants or for intratumoral administration but have limited efficacy as single agents. TargImmune’s platform allows systemic delivery of dsRNA, tailoring the nanoparticles to various solid cancers by simply changing the targeting moiety. Here we describe EGFR-Ta:RNA, which is targeted to tumors that overexpress EGFR. EGFR-Ta:RNA does not inhibit EGFR per se, and therefore activation of alternative or downstream oncogenes is not expected to cause resistance. The mechanism of action of EGFR-targeted Ta:RNA nanoparticles was extensively studied in vitro and in vivo, using confocal imaging, large-scale RNA sequencing studies, Western blot analysis and ELISA assays. In vitro, EGFR-Ta:RNA potency and selectivity were established by comparing cells with high receptor and low receptor expression. Imaging studies showed that the nanoparticles are internalized selectively into EGFR-overexpressing cancer cells by receptor-mediated endocytosis. RNAseq studies revealed: (i) a common transcriptional signature in EGFR-Ta:RNA-treated tumours and cancer cells; (ii) enrichment in pathways related to anti-viral response, inflammation, type I interferon signaling, and NFΚB signaling; (iii) enrichment in genes coding for cytokines. Western blot analysis and ELISA assays confirmed that the mechanism of action of EGFR-Ta:RNA is mediated via the induction of PRR downstream signaling, which results in cytokine secretion, apoptosis and immune cell activation. The efficacy of EGFR-Ta:RNA was demonstrated in multiple animal models, including both xenograft and immunocompetent models. The pharmacodynamic effect, exemplified by target engagement and activation of PRR signaling in tumors, was verified in vivo. In conclusion, TargImmune’s EGFR-Ta:RNA nanoparticles deliver polyIC in a tumor-targeted manner to evoke PRR signaling, inducing targeted tumor cell apoptosis as well as immune cell recruitment and activation against the heterogenous tumor. EGFR-Ta:RNA represents a novel, multimodal treatment approach for EGFR overexpressing cancers. Citation Format: Derrick Broka, Caroline De Feyter, Shoshana Klein, Meera Saxena, Joerg Schreiber, Alexei Shir, Michal Skowicki, Yaakov Benenson, Alexander Levitzki, Cornelia G. Palivan, Esteban Pombo-Villar, Babette Schade, Maya Zigler. Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5799.

Abstract 5235: Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids

Abstract Background: Increasing the immunogenicity of solid tumors may potentiate durable anti-tumor immunity. Targeting innate pattern recognition receptor signaling is a promising strategy to repolarizing the suppressive tumor immune microenvironment (TME). A potential synergistic approach is the addition of cellular therapy. Tumor models expressing high levels of endogenous STING, but where the pathway is not active, were considered for evaluating STING agonism as a potential intervention. Additionally, prior work has demonstrated that STING induction is toxic to T cells; however, natural killer (NK) cells are unaffected, due to their differential regulation of autophagy. Here we investigate the immunomodulatory properties of STING agonism alone and in combination with NK cells using short-term microfluidic culture of fresh patient-derived organotypic tumor spheroids (PDOTS). Methods: Surgical cases from Brigham and Women’s Hospital under an IRB-approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by fluorescent live/dead imaging, immunofluorescence (IF), multiplexed cytokine array, and single cell RNA sequencing (scRNAseq). Baseline immune phenotypes were analyzed by FACS from single cells isolated during tumor sample preparation. Results: Twelve explants were studied. Two specimens were excluded from viability assessment due to excess of fibrotic material and low viability. Using a cut-off of -40%, three cases were characterized as responders (R) and seven as non-responders (NR). We observed significant induction of CXCL10 in all samples treated with STING agonist. IFN-β, IFN-γ, TNF-α, and MIP1-α were also consistently induced by STING agonist treatment. More detailed analysis of two samples by scRNAseq revealed a strong interferon stimulated genes (ISG) signature in STING agonist conditions; the cellular source of CXCL10 secretion in STING agonist treatment was tumor cells and fibroblasts. We also observed profound changes in myeloid biology, indicating that these cells polarized to a more pro-inflammatory and antigen-presenting phenotype with STING agonism. Interestingly, NK-based cell therapy added to PDOTS cultures displayed better persistence and enhanced effector function relative to endogenous NK cells, with and without STING agonism. Conclusions: Using tumor explants and a variety of orthogonal techniques, we investigated the response to STING agonism and NK cellular therapy ex vivo. Our results indicate that STING agonism remodels the TME, creating a more immune-permissive environment. STING agonism induces secretion of chemoattractant CXCL10 by tumor cells and fibroblasts. STING agonism does not abrogate NK cell effector function and therefore, may synergize with NK cell therapies. Citation Format: Patrick H. Lizotte, Elena Ivanova, Sung Park, Nathaniel Spicer, Sophie Kivlehan, Iliana Gjeci, Stefan Kiesgen, Vicky Appleman, LeeAnn Talarico, Michael Y. Tolstorukov, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Combination STING agonist and NK cellular therapy in patient derived organotypic tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5235.

Towards an integrated understanding of inflammatory pathway influence on hematopoietic stem and progenitor cell differentiation.

Recent research highlights that inflammatory signaling pathways such as pattern recognition receptor (PRR) signaling and inflammatory cytokine signaling play an important role in both on-demand hematopoiesis as well as steady-state hematopoiesis. Knockout studies have demonstrated the necessity of several distinct pathways in these processes, but often lack information about the contribution of specific cell types to the phenotypes in question. Transplantation studies have increased the resolution to the level of specific cell types by testing the necessity of inflammatory pathways specifically in donor hematopoietic stem and progenitor cells (HSPCs) or in recipient niche cells. Here, we argue that for an integrated understanding of how these processes occur in vivo and to inform the development of therapies that modulate hematopoietic responses, we need studies that knockout inflammatory signaling receptors in a cell-specific manner and compare the phenotypes caused by knockout in individual niche cells versus HSPCs.

Cell-intrinsic C5a synergizes with Dectin-1 in macrophages to mediate fungal killing

The complement factor C5a is a core effector product of complement activation. C5a, acting through its receptors C5aR1 and C5aR2, exerts pleiotropic immunomodulatory functions in myeloid cells, which is vital for host defense against pathogens. Pattern-recognition receptors (PRRs) are similarly expressed by immune cells as detectors of pathogen-associated molecular patterns. Although there is evidence of cross talk between complement and PRR signaling pathways, knowledge of the full potential for C5a-PRR interaction is limited. In this study, we comprehensively investigated how C5a signaling through C5a receptors can modulate diverse PRR-mediated cytokine responses in human primary monocyte-derived macrophages and observed a powerful, concentration-dependent bidirectional effect of C5a on PRR activities. Unexpectedly, C5a synergized with Dectin-1, Mincle, and STING in macrophages to a much greater extent than TLRs. Notably, we also identified that selective Dectin-1 activation using depleted zymosan triggered macrophages to generate cell-intrinsic C5a, which acted on intracellular and cell surface C5aR1, to help sustain mitochondrial ROS generation, up-regulate TNFα production, and enhance fungal killing. This study adds further evidence to the holistic functions of C5a as a central immunomodulator and important orchestrator of pathogen sensing and killing by phagocytes.

The Role of TRIM Proteins in Antiviral Defense

The Tripartite motif (TRIM) protein family is classified among a cohort of E3 ubiquitin ligase families. A large percentage of TRIM proteins are E3 ubiquitin ligase active and take part in a number of cellular functions. By adding ubiquitin molecules to target proteins via a variety of techniques, it can change those proteins. The important role of the TRIM protein in controlling pattern recognition receptor signaling pathways and the defense mechanisms of mammals against viruses, with a focus on protecting the host from viral infections, has been clarified by recent study. In addition to triggering crucial cell-intrinsic defense mechanisms like autophagy and transcription-dependent antiviral responses, TRIM proteins can also exhibit direct antiviral effects by obstructing particular viral components via a variety of mechanisms. Regrettably, certain viruses have developed efficient mechanisms to evade the antiviral activities exerted by specific TRIM proteins, including SARS-CoV-2. Viruses has the capacity to exploit TRIMs and the ubiquitination process directly, hence enhancing the viral replication cycle and inducing heightened pathogenicity. In this article, I have delved into research that explores the molecular mechanisms responsible for the antiviral effects of TRIM proteins. Additionally, I have examined how TRIM proteins exert their influence on various viral entities, shedding light on their crucial role in the immune response against viral infections.

The entanglement of DNA damage and pattern recognition receptor signaling

Cells are under constant pressure to suppress DNA damage originating from both exogenous and endogenous sources. Cellular responses to DNA damage help to prevent mutagenesis and cell death that arises when DNA damage is either left unrepaired or repaired inaccurately. During the “acute phase” of DNA damage signaling, lesions are recognized, processed, and repaired to restore the primary DNA sequence whilst cell cycle checkpoints delay mitotic progression, cell death and the propagation of errors to daughter cells. Increasingly, there is recognition of a “chronic phase” of DNA damage signaling, exemplified by the secretion of dozens of cytokines days after the inciting damage event. In this review, we focus on the cellular origin of these chronic responses, the molecular pathways that control them and the increasing appreciation for the interconnection between acute and chronic DNA damage responses.

DNA-Dependent Interferon Induction and Lung Inflammation in Bordetella pertussis Infection.

Pertussis, caused by Bordetella pertussis, is a resurgent respiratory disease but the molecular mechanisms underlying pathogenesis are poorly understood. We recently showed the importance of type I and type III interferon (IFN) induction and signaling for the development of lung inflammation in B. pertussis-infected mouse models. Classically, these IFNs are induced by signaling through a variety of pattern recognition receptors (PRRs) on host cells. Here, we found that the PRR signaling adaptor molecules MyD88 and TRIF contribute to IFN induction and lung inflammatory pathology during B. pertussis infection. However, the PRRs Toll-like receptors (TLR) 3 and TLR4, which signal through TRIF and MyD88, respectively, played no role in IFN induction. Instead, the DNA-sensing PRRs, TLR9 and STING, were important for induction of type I/III IFN and promotion of inflammatory pathology, indicating that DNA is a major inducer of lung IFN responses in B. pertussis infection. These results increase our understanding of this host-pathogen interaction and identify potential targets for host-directed therapies to reduce B. pertussis-mediated pathology.

Chemical profiling and mechanistic studies of Zhi-Shang-Feng granules against influenza virus by high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high-resolution mass spectrometry in combination with network pharmacology analysis.

Zhi-Shang-Feng Granules are used in the clinical treatment of influenza to relieve headaches, chills and fever, bronchitis, nasal congestion, neuralgia and other symptoms. To decipher the components responsible for therapeutic effects of Zhi-Shang-Feng g ranules against influenza virus, an analytical method based on high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high resolution mass spectrometry was developed and the chemical profile of Zhi-Shang-Feng granules was characterized. Then, the identified components were used to conduct network pharmacological analysis and determine the potential mechanism of Zhi-Shang-Feng Granules. As a result, 177 compounds were putatively identified through comprehensive analysis by liquid chromatography coupled with high-resolution mass spectrometry, of which 23 compounds were unambiguously confirmed with reference standards. Components in Zhi-Shang-Feng Granules were found to specifically act on different enzymes, G-protein-coupled receptors, ion channels and transporters in the immune, endocrine, nervous, and circulatory systems. The potential mechanism was related to several biological processes, including cell growth and death, pattern recognition receptor signalling, signalling by interleukins, and lipid metabolism. The combination of chemical profile characterization and network construction provided useful insight into the overall chemical composition of Zhi-Shang-Feng granules and revealed their potential anti-infection, anti-inflammatory and immunoregulatory mechanisms against influenza virus infected disease.

Beyond natural biology: rewiring cellular networks to study innate immunity.

Within immune cells, microbial and self-ligands trigger pattern recognition receptors (PRRs) to nucleate and activate the signaling organelles of the immune system. Much work in this area has derived from observational biology of natural innate immune signaling. More recently, synthetic biology approaches have been used to rewire and study innate immune networks. By utilizing controllable chemical or optogenetic inputs, rearranging protein building blocks, or engineering signal recording circuits, synthetic biology-based techniques complement and inform studies of natural immune pathway operation. In this review, we describe recent synthetic biology-based approaches that have uncovered new insights into PRR signaling, virus-host interactions, and systemic cytokine responses.

The immunosuppressive activity of myeloid-derived suppressor cells in murine Paracoccidioidomycosis relies on Indoleamine 2,3-dioxygenase activity and Dectin-1 and TLRs signaling

Paracoccidioidomycosis (PCM) is a systemic mycosis with a high incidence in Latin America. Prior studies have demonstrated the significance of the enzyme Indoleamine 2,3-dioxygenase (IDO-1) in the immune regulation of PCM as well as the vital role of myeloid-derived suppressor cells (MDSCs) in moderating PCM severity. Additionally, Dectin-1 and Toll-Like Receptors (TLRs) signaling in cancer, infection, and autoimmune diseases have been shown to impact MDSC-IDO-1+ activity. To expand our understanding of MDSCs and the role of IDO-1 and pattern recognition receptors (PRRs) signaling in PCM, we generated MDSCs in vitro and administered an IDO-1 inhibitor before challenging the cells with Paracoccidioides brasiliensis yeasts. By co-culturing MDSCs with lymphocytes, we assessed T-cell proliferation to examine the influence of IDO-1 on MDSC activity. Moreover, we utilized specific antibodies and MDSCs from Dectin-1, TLR4, and TLR2 knockout mice to evaluate the effect of these PRRs on IDO-1 production by MDSCs. We confirmed the importance of these in vitro findings by assessing MDSC-IDO-1+ in the lungs of mice following the fungal infection. Taken together, our data show that IDO-1 expression by MDSCs is crucial for the control of T-cell proliferation, and the production of this enzyme is partially dependent on Dectin-1, TLR2, and TLR4 signaling during murine PCM.

Multiple Porcine Innate Immune Signaling Pathways Are Involved in the Anti-PEDV Response.

Porcine epidemic diarrhea virus (PEDV) has caused great damage to the global pig industry. Innate immunity plays a significant role in resisting viral infection; however, the exact role of innate immunity in the anti-PEDV response has not been fully elucidated. In this study, we observed that various porcine innate immune signaling adaptors are involved in anti-PEDV (AJ1102-like strain) activity in transfected Vero cells. Among these, TRIF and MAVS showed the strongest anti-PEDV activity. The endogenous TRIF, MAVS, and STING were selected for further examination of anti-PEDV activity. Agonist stimulation experiments showed that TRIF, MAVS, and STING signaling all have obvious anti-PEDV activity. The siRNA knockdown assay showed that TRIF, MAVS, and STING are also all involved in anti-PEDV response, and their remarkable effects on PEDV replication were confirmed in TRIF-/-, MAVS-/- and STING-/- Vero cells via the CRISPR approach. For further verification, the anti-PEDV activity of TRIF, MAVS, and STING could be reproduced in porcine IPEC-DQ cells treated with siRNAs. In summary, this study reveals that multiple pattern-recognition receptor (PRR) signaling pathways of porcine innate immunity play an important role in the anti-PEDV infection, providing new and useful antiviral knowledge for prevention and control of PEDV spreading.

Tinospora cordifolia Miers enhances the immune response in mice immunized with JEV-vaccine: A network pharmacology and experimental approach

BackgroundTinospora cordifolia Miers. (TC) (Giloya/Guduchi) is a native Indian herb, reported for its wide array of medicinal activities including immunomodulatory activity. However, the exact pharmacological mechanism of TC as an immunomodulatory agent remains unclear. Central to this, to the best of our knowledge, no study has explored the immunoadjuvant potential of TC in response to the Japanese encephalitis (JE) vaccines. PurposeThe study aims to explore the immunoadjuvant potential of TC ethanolic extract in response to the JE vaccine and illustrates its potential mechanism of immunomodulation using an integrated approach of network pharmacology and in-vivo experimental study. Study design and methodsInitially, the extract was prepared and the components of TC were identified through high-resolution liquid chromatography mass spectrometry (HR-LC/MS). The compounds were then screened for network pharmacology analysis. Next, the drug and disease targets were identified and the network was constructed using Cytoscape 3.7.2 to obtain different signalling pathways of TC in JEV. We then evaluated the immunoadjuvant potential of TC ethanolic extract in mice immunized with inactivated JE vaccine (SA-14–14–2 strain). BALB/c mice were supplemented with TC extract (30 and 100 mg/kg, i.g.), daily for 56 days, marked with immunization on 28th day of the study, by JE vaccine. Blood was collected for flow cytometry and haematological analysis (total and differential cell counts). The surface expression of immune-cell markers (CD3+, CD4+, CD19+, CD11c+, CD40+) were evaluated on day 0 (pre-immunization), day 14 and 28 post-immunization. Additionally, inflammatory cytokines (IFN-γ+/IL-17A+) were evaluated post-14 and 28 days of immunization. ResultsThe HR-LC/MS analysis identified the presence of glycosides, terpenoids, steroids and alkaloids in the TC extract. Through network analysis, 09 components and 166 targets were obtained, including pathways that involve toll-like receptor signalling, pattern-recognition receptor signalling, cytokine receptor and cytokine mediated signalling, etc. The in-vivo results showed that preconditioning with TC ethanolic extract significantly elevated the haematological variables (leucocyte count) as well as the surface expression of CD markers (B and T cell subsets) on day 0 (pre-immunization), day 14 and 28 post-immunization. Furthermore, preconditioning of TC demonstrated a dose-dependant augmentation of immune cells (CD3+, CD4+, CD19+, CD11c+) and inflammatory cytokines (IFN-γ+/IL-17A+) on day 14 and 28 post-immunization when compared to vaccine alone group. ConclusionResults showed that preconditioning with TC extract before immunization might play a potential role in enhancing the cell-mediated as well as humoral immunity. Altogether, the combinatorial approach of network pharmacology and in-vivo animal experimentation demonstrated the immunoadjuvant potential of TC in response to JEV vaccine.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

The Extract of Scutellaria baicalensis Attenuates the Pattern Recognition Receptor Pathway Activation Induced by Influenza A Virus in Macrophages.

The dual strategy of inhibiting the viral life cycle and reducing the host inflammatory response should be considered in the development of therapeutic drugs for influenza A virus (IAV). In this study, an extract of Scutellaria baicalinase (SBE) containing seven flavonoids was identified to exert both antiviral and anti-inflammatory effects in macrophages infected with IAV. We performed transcriptome analysis using high-throughput RNA sequencing and identified 315 genes whose transcription levels were increased after IAV infection but were able to be decreased after SBE intervention. Combined with Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, these genes were mainly involved in TLR3/7/8, RIG-I/MDA5, NLRP3 and cGAS pattern recognition receptor (PRR)-mediated signaling pathways. SBE inhibited the transcription of essential genes in the above pathways and nuclear translocation of NF-κB p65 as confirmed by RT-qPCR and immunofluorescence, respectively, indicating that SBE reversed PR8-induced over-activation of the PRR signaling pathway and inflammation in macrophages. This study provides an experimental basis for applying Scutellaria baicalensis and its main effects in the clinical treatment of viral pneumonia. It also provides novel targets for screening and developing novel drugs to prevent and treat IAV infectious diseases.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

The manipulation of cell suspensions from zebrafish intestinal mucosa contributes to understanding enteritis.

Although zebrafish are commonly used to study intestinal mucosal immunity, no dedicated procedure for isolating immune cells from zebrafish intestines is currently available. A speedy and simple operating approach for preparing cell suspension from mucosa has been devised to better understanding of intestinal cellular immunity in zebrafish. The mucosal villi were separated away from the muscle layer by repeated blows. The complete deprivation of mucosa was done and evidenced by HE and qPCR results. Higher expression of both innate (mpeg1, mpx, and lck) and adaptive immune genes (zap70, blnk, foxp3a, and foxp3b) was revealed compared to cells obtained by typical mesh rubbing. The cytometric results also revealed that the tested operation group had a higher concentration and viability. Further, fluorescent-labelled immune cells from 3mo Tg(lyz:DsRED2), Tg(mpeg1:EGFP), Tg(Rag2:DsRED), and Tg(lck:EGFP), were isolated and evaluated for the proportion, and immune cells' type could be inferred from the expression of marker genes. The transcriptomic data demonstrated that the intestinal immune cell suspension made using the new technique was enriched in immune-related genes and pathways, including il17a/f, il22, cd59, and zap70, as well as pattern recognition receptor signaling and cytokine-cytokine receptor interaction. In addition, the low expression of DEG for the adherent and close junctions indicated less muscular contamination. Also, lower expression of gel-forming mucus-associated genes in the mucosal cell suspension was consistent with the current less viscous cell suspension. To apply and validate the developed manipulation, enteritis was induced by soybean meal diet, and immune cell suspensions were analyzed by flow cytometry and qPCR. The finding that in enteritis samples, there was inflammatory increase of neutrophils and macrophages, was in line with upregulated cytokines (il8 and il10) and cell markers (mpeg1 and mpx). As a result, the current work created a realistic technique for studying intestinal immune cells in zebrafish. The immune cells acquired may aid in further research and knowledge of intestinal illness at the cellular level.

Oncolytic Rodent Protoparvoviruses Evade a TLR- and RLR-Independent Antiviral Response in Transformed Cells.

The oncolytic rodent protoparvoviruses (PVs) minute virus of mice (MVMp) and H-1 parvovirus (H-1PV) are promising cancer viro-immunotherapy candidates capable of both exhibiting direct oncolytic activities and inducing anticancer immune responses (AIRs). Type-I interferon (IFN) production is instrumental for the activation of an efficient AIR. The present study aims at characterizing the molecular mechanisms underlying PV modulation of IFN induction in host cells. MVMp and H-1PV triggered IFN production in semi-permissive normal mouse embryonic fibroblasts (MEFs) and human peripheral blood mononuclear cells (PBMCs), but not in permissive transformed/tumor cells. IFN production triggered by MVMp in primary MEFs required PV replication and was independent of the pattern recognition receptors (PRRs) Toll-like (TLR) and RIG-like (RLR) receptors. PV infection of (semi-)permissive cells, whether transformed or not, led to nuclear translocation of the transcription factors NFĸB and IRF3, hallmarks of PRR signaling activation. Further evidence showed that PV replication in (semi-)permissive cells resulted in nuclear accumulation of dsRNAs capable of activating mitochondrial antiviral signaling (MAVS)-dependent cytosolic RLR signaling upon transfection into naïve cells. This PRR signaling was aborted in PV-infected neoplastic cells, in which no IFN production was detected. Furthermore, MEF immortalization was sufficient to strongly reduce PV-induced IFN production. Pre-infection of transformed/tumor but not of normal cells with MVMp or H-1PV prevented IFN production by classical RLR ligands. Altogether, our data indicate that natural rodent PVs regulate the antiviral innate immune machinery in infected host cells through a complex mechanism. In particular, while rodent PV replication in (semi-)permissive cells engages a TLR-/RLR-independent PRR pathway, in transformed/tumor cells this process is arrested prior to IFN production. This virus-triggered evasion mechanism involves a viral factor(s), which exert(s) an inhibitory action on IFN production, particularly in transformed/tumor cells. These findings pave the way for the development of second-generation PVs that are defective in this evasion mechanism and therefore endowed with increased immunostimulatory potential through their ability to induce IFN production in infected tumor cells.

Ellagitannin Punicalagin Disrupts the Pathways Related to Bacterial Growth and Affects Multiple Pattern Recognition Receptor Signaling by Acting as a Selective Histone Deacetylase Inhibitor

Punicalagin (PA) is a key ellagitannin abundant in pomegranate with wide-ranging biological activities. In this study, we examined the biological processes by which PA regulates bacterial growth and inflammation in human cells using multiomics and molecular docking approaches. PA promoted macrophage-mediated bacterial killing and inhibited the growth of Citrobacter rodentium by inducing a distinct metabolome pattern. PA acted as a selective regulator of histone deacetylases (HDACs) and affected 37 pathways in macrophages, including signaling mediated by pattern recognition receptors, such as Toll-like and NOD-like receptors. In silico simulation showed that PA can bind with high affinity to HDAC7. PA downregulated HDAC7 at both mRNA and protein levels and resulted in a decrease in the level of histone 3 lysine 27 acetylation. Our findings provide evidence that PA exerts its biological effects via multiple pathways, which can be exploited in the development of this bioactive food ingredient for disease management.

Autophagy as a dual-faced host response to viral infections.

Autophagy selectively degrades viral particles or cellular components, either facilitating or inhibiting viral replication. Conversely, most viruses have evolved strategies to escape or exploit autophagy. Moreover, autophagy collaborates with the pattern recognition receptor signaling, influencing the expression of adaptor molecules involved in the innate immune response and regulating the expression of interferons (IFNs). The intricate relationship between autophagy and IFNs plays a critical role in the host cell defense against microbial invasion. Therefore, it is important to summarize the interactions between viral infections, autophagy, and the host defense mechanisms against viruses. This review specifically focuses on the interactions between autophagy and IFN pathways during viral infections, providing a comprehensive summary of the molecular mechanisms utilized or evaded by different viruses.