Cellular Innate Immune Responses Research Articles

Predicting the immunomodulatory activity of probiotic lactic acid bacteria using supervised Machine Learning in a Cornu aspersum snail model

In the process of screening for probiotic strains, there are no clearly established bacterial phenotypic markers which could be used for the prediction of their in vivo mechanism of action. In this work, we demonstrate for the first time that Machine Learning (ML) methods can be used for accurately predicting the in vivo immunomodulatory activity of probiotic strains based on their cell surface phenotypic features using a snail host-microbe interaction model. A broad range of snail gut presumptive probiotics, including 240 new lactic acid bacterial strains (Lactobacillus, Leuconostoc, Lactococcus, and Enterococcus), were isolated and characterized based on their capacity to withstand snails’ gastrointestinal defense barriers, such as the pedal mucus, gastric mucus, gastric juices, and acidic pH, in association with their cell surface hydrophobicity, autoaggregation, and biofilm formation ability. The implemented ML pipeline predicted with high accuracy (88%) strains with a strong capacity to enhance chemotaxis and phagocytic activity of snails’ hemolymph cells, while also revealed bacterial autoaggregation and cell surface hydrophobicity as the most important parameters that significantly affect host immune responses. The results show that ML approaches may be useful to derive a predictive understanding of host-probiotic interactions, while also highlighted the use of snails as an efficient animal model for screening presumptive probiotic strains in the light of their interaction with cellular innate immune responses.

Pharmacological activation of TLR7 exerts inhibition on the replication of EV-D68 in respiratory cells.

The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.

Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

RNA-Seq of an LPS-Induced Inflammation Model Reveals Transcriptional Profile Patterns of Inflammatory Processes.

The LPS-induced inflammation model is widely used for studying inflammatory processes due to its cost-effectiveness, reproducibility, and faithful representation of key hallmarks. While researchers often validate this model using clinical cytokine markers, a comprehensive understanding of gene regulatory mechanisms requires extending investigation beyond these hallmarks. Our study leveraged multiple whole-blood bulk RNA-seq datasets to rigorously compare the transcriptional profiles of the well-established LPS-induced inflammation model with those of several human diseases characterized by systemic inflammation. Beyond conventional inflammation-associated systems, we explored additional systems indirectly associated with inflammatory responses (i.e., ISR, RAAS, and UPR) using a customized core inflammatory gene list. Our cross-condition-validation approach spanned four distinct conditions: systemic lupus erythematosus (SLE) patients, dengue infection, candidemia infection, and staphylococcus aureus exposure. This analysis approach, utilizing the core gene list aimed to assess the model's suitability for understanding the gene regulatory mechanisms underlying inflammatory processes triggered by diverse factors. Our analysis resulted in elevated expressions of innate immune-associated genes, coinciding with suppressed expressions of adaptive immune-associated genes. Also, upregulation of genes associated with cellular stresses and mitochondrial innate immune responses underscored oxidative stress as a central driver of the corresponding inflammatory processes in both the LPS-induced and other inflammatory contexts.

Pre-Infection Innate Immunity Attenuates SARS-CoV-2 Infection and Viral Load in iPSC-Derived Alveolar Epithelial Type 2 Cells.

A large portion of the heterogeneity in coronavirus disease 2019 (COVID-19) susceptibility and severity of illness (SOI) remains poorly understood. Recent evidence suggests that SARS-CoV-2 infection-associated damage to alveolar epithelial type 2 cells (AT2s) in the distal lung may directly contribute to disease severity and poor prognosis in COVID-19 patients. Our in vitro modeling of SARS-CoV-2 infection in induced pluripotent stem cell (iPSC)-derived AT2s from 10 different individuals showed interindividual variability in infection susceptibility and the postinfection cellular viral load. To understand the underlying mechanism of the AT2's capacity to regulate SARS-CoV-2 infection and cellular viral load, a genome-wide differential gene expression analysis between the mock and SARS-CoV-2 infection-challenged AT2s was performed. The 1393 genes, which were significantly (one-way ANOVA FDR-corrected p ≤ 0.05; FC abs ≥ 2.0) differentially expressed (DE), suggest significant upregulation of viral infection-related cellular innate immune response pathways (p-value ≤ 0.05; activation z-score ≥ 3.5), and significant downregulation of the cholesterol- and xenobiotic-related metabolic pathways (p-value ≤ 0.05; activation z-score ≤ -3.5). Whilst the effect of post-SARS-CoV-2 infection response on the infection susceptibility and postinfection viral load in AT2s is not clear, interestingly, pre-infection (mock-challenged) expression of 238 DE genes showed a high correlation with the postinfection SARS-CoV-2 viral load (FDR-corrected p-value ≤ 0.05 and r2-absolute ≥ 0.57). The 85 genes whose expression was negatively correlated with the viral load showed significant enrichment in viral recognition and cytokine-mediated innate immune GO biological processes (p-value range: 4.65 × 10-10 to 2.24 × 10-6). The 153 genes whose expression was positively correlated with the viral load showed significant enrichment in cholesterol homeostasis, extracellular matrix, and MAPK/ERK pathway-related GO biological processes (p-value range: 5.06 × 10-5 to 6.53 × 10-4). Overall, our results strongly suggest that AT2s' pre-infection innate immunity and metabolic state affect their susceptibility to SARS-CoV-2 infection and viral load.

Dual RNA-seq Analysis of Patients' Cells and Viral Genome After Measles Infection.

During the infection of a host cell by an infectious agent, a series of gene expression changes occurs as a consequence of host-pathogen interactions. Unraveling this complex interplay is the key for understanding of microbial virulence and host response pathways, thus providing the basis for new molecular insights into the mechanisms of pathogenesis and the corresponding immune response. Dual RNA sequencing (dual RNA-seq) has been developed to simultaneously determine pathogen and host transcriptomes enabling both differential and coexpression analyses between the two partners as well as genome characterization in the case of RNA viruses. Here, we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on - but not restricted to - measles virus (MeV) as a pathogen of interest. The application of dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the structure of the viral RNA genome and on cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

USP18 promotes innate immune responses and apoptosis in influenza A virus-infected A549 cells via cGAS-STING pathway

Influenza A virus (IAV) can infect respiratory epithelial cells where it replicates, triggers cellular innate immune responses, and even induces cell apoptosis. Ubiquitin-specific peptidase 18 (USP18) was reported to be associated with IAV replication and immune response homeostasis. Therefore, this study aimed to investigate the role of USP18 in IAV-infected lung epithelial cells. The cell viability was determined by the CCK-8 method. Viral titers were quantified by standard plaque assay. Innate immune response-associated cytokines were detected by RT-qPCR and ELISA and cell apoptosis was assessed by flow cytometry. The results showed that overexpression of USP18 promoted viral replication, innate immune factor secretion and apoptosis in IAV-infected A549 cells. Mechanistically, USP18 reduced cGAS degradation by decreasing its K48-linked ubiquitination to promote IAV-induced cGAS-STING pathway activation. In conclusion, USP18 is a pathological mediator of IAV in lung epithelial cells.

#3636 INHIBITION OF THE STING PATHWAY AMELIORATES EXPERIMENTAL CRESCENTIC GLOMERULONEPHRITIS

Abstract Background and Aims Rapidly progressive glomerulonephritis/crescentic glomerulonephritis (RPGN/cGN) may compromise renal function in the short or medium term thus evolving into acute kidney injury (AKI) or chronic kidney disease. Innate and adaptive immune responses are placed at the epicentre of cGN. However, molecular pathways involved in the activation of these responses are by far less known. Sting is an innate immune DNA-sensing involved in promoting kidney injury and fibrosis with unknown participation in RPGN/cGN. Method We investigated whether Sting pathway activation contributes to the development and progression of RPGN/cGN in a preclinical model of nephrotoxic nephritis (NTN) induced by an anti-glomerular basal membrane serum. Sting expression as well as humoral and cellular innate immune responses, histological injury and functional responses in the kidney were studied in wild-type and Sting-/- mice. Results Sting was found expressed at higher gene and protein levels and located in tubulointerstitial and intraglomerular and periglomerular areas and tubules in kidneys from NTN wild-type mice compared to non-diseased control animals. Sting deficiency significantly decreased NTN-induced cytokine/chemokine synthesis and hence the intrarenal transit of both mononuclear phagocytes and overall or specific T-cell subsets. Sting-deficient mice also displayed less extensive histological affection, namely a fewer number of glomerular crescents and associated lesions (sclerosis, endocapillary hypercellularity/sinaquiae), and tubular injury. The presence of TUNEL-positive dead cells was also significantly reduced in Sting-/- mice. Consistent with these findings, Sting absence significantly diminished NTN-increased urea and creatinine plasma levels. Conclusion Sting inhibition positively impacted several well-characterized cGN-associated pathogenic events thus ameliorating the outcomes of the disease. These results identified Sting antagonism as a potential therapy to help combat cGN.

Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease.

131 Alveolar Macrophage Degranulation Initiates the Spatially Restricted Innate Immune Response During Ventilator-Assisted Nanoparticle Inhalation

Abstract To visualize and measure in real-time the cellular pulmonary innate immune response elicited by inhaled NPs, we apply state-of-the-art intravital microscopy on the alveolar region of the murine lung. Fluorescent carboxyl-quantum-dot-NPs (deposited dose of 16 cm2/g) applied via ventilator-assisted inhalation accumulated within seconds as distinct fluorescent spots at the alveolar walls. Already at 30min, cQD-NPs elicited an increase in microvascular neutrophil numbers as compared to controls, which subsequently transmigrated into the alveolar space. This early immune response was not specific to cQD-NPs, since a comparable increase in neutrophil numbers was also observed upon inhalation of a bioequivalent dose of carbon black NPs, a typical component of urban air pollution. AMs actively accumulated cQD-NPs over time, starting few minutes after inhalation. Significantly more neutrophils were attracted in proximity to cQD-NP-laden AMs, as compared to NP-free AMs or control areas with epithelial associated cQD-NP. Prior airway application of a cellular degranulation inhibitor prevented cQD-NP and CNP induced neutrophil recruitment at 2h and 24 h after inhalation. Inhibition of Fcγ-receptor mediated AM cQD-NP uptake by prior application of anti-CD64 mAbs or neutralizing of TNFα by anti-TNFα mAbs in the airspace, abolished this early immune response. Importantly, initial ImageStream flow cytometry analysis of BAL samples indicated an increase of macrophage-derived microvesicles upon NP inhalation. Taken together, our data links NP-induced AM degranulation and cytokine release to the rapid and site-specific recruitment of neutrophils, suggesting this process to be a key event in mounting the immune response upon NP inhalation.

Unexpected Need of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity for Signaling by Intracellular Pattern Recognition Receptors of Nucleic Acids.

Many pattern recognition receptors in mammalian cells initiate signaling processes that culminate in mounting an innate protective response mediated by induced synthesis of a large number of proteins including type I interferons and other cytokines. Many of these receptors are not located on the plasma membrane but on the membranes of intracellular organelles such as endosomes, mitochondria, and the endoplasmic reticulum; they primarily recognize microbial or cellular nucleic acids. In the course of biochemical analyses of the signaling pathways triggered by these receptors, we discovered that they require tyrosine phosphorylation by the protein kinase activity of the epidermal growth factor receptor (EGFR), which is located not only on the plasma membrane but also on the intracellular membranes. Here, we discuss how specific members of this family of receptors, such as TLR3, TLR9, or STING, interact with EGFR and other protein tyrosine kinases and what are the functional consequences of their post-translational modifications. The article highlights an unexpected functional link between a growth factor receptor and cellular innate immune response.

Antiviral activities of hemp cannabinoids.

Hemp is an understudied source of pharmacologically active compounds and many unique plant secondary metabolites including more than 100 cannabinoids. After years of legal restriction, research on hemp has recently demonstrated antiviral activities in silico, in vitro, and in vivo for cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), and several other cannabinoids against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), human immunodeficiency virus (HIV), and γ-herpes viruses. Mechanisms of action include inhibition of viral cell entry, inhibition of viral proteases, and stimulation of cellular innate immune responses. The anti-inflammatory properties of cannabinoids are also under investigation for mitigating the cytokine storm of COVID-19 and controlling chronic inflammation in people living with HIV. Retrospective clinical studies support antiviral activities of CBD, Δ9-THC, and cannabinoid mixtures as do some prospective clinical trials, but appropriately designed clinical trials of safety and efficacy of antiviral cannabinoids are urgently needed.

Intestinal colonization with Candida auris and mucosal immune response in mice treated with cefoperazone oral antibiotic.

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with Candida albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. A significant increase in Candida-specific IgA was observed in the serum of C. auris but not in the C. albicans infected mice. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

Enterobacter asburiae E7, a Novel Potential Probiotic, Enhances Resistance to Aeromonas veronii Infection via Stimulating the Immune Response in Common Carp (Cyprinus carpio).

Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1β, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-β at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls (54%; P < 0.01). Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic. IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety.

Coronaviruses, Lysosomes, and Secondary Bacterial Infections: Coronaviruses Outsmart the Host.

Lysosomes are key organelles that contribute to homeostatic functions such as autophagy-mediated recycling of cellular components and innate immune response through phagocytosis-mediated pathogen killing during infections. Viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has developed unique adaptation to not only avoid lysosome-mediated destruction but also actively utilize lysosomal machinery to both enter and exit cells. To survive the highly hostile lysosomal environment, coronaviruses deacidify the lysosomes, potentially by manipulating H+ ion exchange across the lysosomal lumen, ensuring coronavirus survival. At the same time, this deacidification not only impairs cellular homeostatic functions such as autophagy but also renders the host susceptible to secondary bacterial infections. Furthermore, lysosomal enzymes promote extensive cell death and tissue damage during secondary bacterial infections. Thus, targeting lysosomal pathways provide a great opportunity to limit both viral replication and subsequent negative impact on host immunity against secondary bacterial infections.

Effects of metacercariae of Prosorhynchoides sp. (Trematoda: Bucephalidae) on the swimming ability and blood parameters of the intertidal fish Girella laevifrons (Osteichthyes: Kyphosidae)

Parasites may have various effects on their host's health. It is important to determine the type of the effects but also to decipher the physiological mechanisms underlying a parasitic infection. In this study, swimming ability (by a fixed swim speed test) and haematological analyses (by blood parameters) were compared between juvenile fish Girella laevifrons non-parasitised and parasitised with metacercariae of Prosorhynchoides sp. Fish were infested in the laboratory using natural parasitised mussels. Aerobic swimming time, total erythrocyte and leukocyte counts were compared among four groups of fish: non-parasitised (NP), non-parasitised but manipulated (NPM), parasitised 3 days (PT1), and 10 days (PT2) post-infection. Differentiated haematological components were also compared among NP, PT1 and PT2 fish groups. Parasitised fish (PT1 and PT2) swam between 32% and 55% less time than unparasitised fish. Erythrocyte counts were lower in PT1, indicating anemia, and total leukocyte counts were higher in PT1, showing immune action. Macrophages and basophils were observed only in parasitised fish, independent of the post-infection time. There was a negative correlation between swimming time and leukocyte counts. Increased leukocytes in the blood indicate activation of the innate cellular immune response, which may be metabolically costly for the host. Moreover, the anemia in parasitised fish would reduce the oxygen transport necessary to carry out aerobic exercise, reducing the swimming capacity. In fish several days post-infection (PT2), the erythrocytes count reached normal values, and some kinds of leukocytes decreased in comparison to fish PT1. This suggests that parasitised fish with metacercariae were recovering.

Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes

ABSTRACT Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration. Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1β, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.

Activation of innate immune cGAS-STING pathway contributes to Alzheimer's pathogenesis in 5×FAD mice.

cGAS senses microbial and host-derived double-stranded DNA in cytoplasm to trigger cellular innate immune response in a STING-dependent manner; however, it remains unknown whether the cGAS-STING pathway in innate immunity contributes to Alzheimer's disease (AD). Here we demonstrated the detectable binding of the cGAS double-stranded DNA in cytoplasm and the activation of the microglial cGAS-STING pathway in brains of human AD and aged mice. Cgas-/-;5×FAD mice were largely protected from cognitive impairment, amyloid-β pathology, neuroinflammation and other sequelae associated with AD. Furthermore, Cgas deficiency in microglia inhibited a neurotoxic A1 astrocytic phenotype and thus alleviated oligomeric amyloid-β peptide-induced neurotoxicity. Finally, administration of STING inhibitor H-151 potently suppressed the activation of the cGAS-STING pathway and ameliorated AD pathogenesis in 5×FAD mice. In conclusion, our present study has identified a critical molecular link between innate immunity and AD and suggests that therapeutic targeting of the cGAS-STING pathway activity might effectively interfere with the progression of AD.

Seeing the T cell Immunity of SARS-CoV-2 and SARS-CoV: Believing the Epitope-Oriented Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 stimulated vigorous research efforts in immunology and vaccinology. In addition to innate immune responses, both virus-specific humoral and cellular immune responses are of importance for viral clearance. T cell epitopes play a central role in T cell-based immune responses. Herein, we summarized the peptide/major histocompatibility complex (pMHC) structures of the SARS-CoV-2-derived T cell epitopes available in the Protein Data Bank (PDB) and proposed the challenge and opportunities for using of T cell epitopes in future vaccine development efforts. A total of 27 SARS-CoV-2 related pMHC structures and five complexes with T cell receptors were retrieved. The peptides are mainly distributed on spike (S), nucleocapsid (N), and ORF1ab proteins. Most peptides are conserved among variants of concerns (VOCs) for SARS-CoV-2, except for several mutated peptides located in the S protein. The structures of human leukocyte antigen (HLA) complexed with seven epitopes derived from SARS-CoV were also retrieved, which showed a potential cross T cell immunity with SARS-CoV-2. Structural studies of antigenic peptides from SARS-CoV-2 and SARS-CoV help to visualize the processes and the mechanisms of cross T cell immunity. T cell epitope-oriented vaccines are potential next-generation vaccines for SARS-CoV-2, which are worthy of further investigation.

Susceptibilities of CNS Cells towards Rabies Virus Infection Is Linked to Cellular Innate Immune Responses.

Rabies is caused by neurotropic rabies virus (RABV), contributing to 60,000 human deaths annually. Even though rabies leads to major public health concerns worldwide, we still do not fully understand factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and immune responses of CNS cell types to infection with two RABV strains, Tha and its attenuated variant Th2P-4M, mutated on phospho- (P-protein) and matrix protein (M-protein). We demonstrate that RABV replicates in human stem cell-derived neurons and astrocytes but fails to infect human iPSC-derived microglia. Additionally, we observed major differences in transcription profiles and quantification of intracellular protein levels between antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, and LIF), and microglia (CCL5, CXCL10, ISG15, MX1, and IL6) upon Tha infection. We also show that P- and M-proteins of Tha mediate evasion of NF-κB- and JAK-STAT-controlled antiviral host responses in neuronal cell types in contrast to glial cells, potentially explaining the strong neuron-specific tropism of RABV. Further, Tha-infected astrocytes and microglia protect neurons from Tha infection via a filtrable and transferable agent. Overall, our study provides novel insights into RABV tropism, showing the interest in studying the interplay of CNS cell types during RABV infection.