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Frontiers in Immunology 2023Type I interferonopathies are a heterogenic group of rare diseases associated with an increase in type I interferon (IFN). The main challenge for the study of Type I...
Type I interferonopathies are a heterogenic group of rare diseases associated with an increase in type I interferon (IFN). The main challenge for the study of Type I interferonopathies is the lack of a well-founded animal model to better characterize the phenotype as well as to perform fast and large drug screenings to offer the best treatment options. In this study, we report the development of a transgenic zebrafish model of Type I interferonopathy overexpressing carrying the mutation p.Arg742His (), corresponding to the human mutation p.Arg779His. RNA sequence analysis from larvae revealed a systemic inflammation and IFN signature upon a suboptimal poly I:C induction compared with wild-type larvae, confirming the phenotype observed in patients suffering from Type I interferonopathies. More interestingly, the phenotype was manifested in the zebrafish inflammation and Type I IFN reporters and , respectively, making this zebrafish model suitable for future high-throughput chemical screening (HTS). Using the unique advantages of the zebrafish model for gene editing, we have generated knocked down for and , which completely abrogated the Poly I:C induction and activation of the GFP of the reporters. Finally, we used an FDA-approved drug, Baricitinib (Jak1/Jak2 inhibitor), which was able to reduce the inflammation and the ISG expression. Our results demonstrate the potential of this model to further understand AGS pathological mechanisms and to identify novel therapeutic drugs by HTS.
Topics: Animals; Humans; Inflammation; Interferon Type I; Poly I; Zebrafish; Interferon-Induced Helicase, IFIH1
PubMed: 38077314
DOI: 10.3389/fimmu.2023.1294766 -
Brain, Behavior, and Immunity Jan 2024To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal...
To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4β7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1β and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4α4β7, unstimulated and stimulated IL-17A and stimulated IFN-γ lymphocytes in MLN and CD4α4β7 unstimulated and stimulated IL-17A and stimulated IFN-γ lymphocytes in the spleen. Treatment with anti-α4β7 mAb in EIA male mice was associated with colonic tissue IL-1β, and IL-17A mRNA content and percentage of CD4 IL-17A and IFN-γ lymphocytes in MLN and spleens comparable to control mice. The anti-α4β7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1β and decreased CD68 and TGF-β mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4β7 mAb treatment restored the expression of BDNF, TGF-β and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4β7+) lymphocytes expressing IL-17A.
Topics: Humans; Adult; Pregnancy; Female; Male; Mice; Animals; Interleukin-17; Autism Spectrum Disorder; Brain-Derived Neurotrophic Factor; Integrins; Antibodies, Monoclonal; Transforming Growth Factor beta; RNA, Messenger
PubMed: 37793488
DOI: 10.1016/j.bbi.2023.09.024 -
Frontiers in Immunology 2023The cultured can meet the market demand in the context of the decline of wild resources, but the disease in the high-density culture process also deserves attention....
INTRODUCTION
The cultured can meet the market demand in the context of the decline of wild resources, but the disease in the high-density culture process also deserves attention. Therefore, understanding the immune regulation mechanisms of will be the basis for obtaining high benefits in artificial culture.
METHODS
To explore the viral response mechanism of , RNA-seq was applied to identify the transcriptomic changes of the liver and spleen in by poly (I:C) stress.
RESULTS
The DEGs (liver: 2186 to 3123; spleen 1542 to 2622) and up-regulated genes (liver: 1231 to 1776; spleen 769 to 1502) in the liver and spleen increased with the prolongation (12h to 48h) of poly (I:C)-stimulation time. This means needs to mobilize more functional genes in response to longer periods of poly (I:C)-stimulation. Despite the responses of to poly (I:C) showed tissue-specificity, we hypothesized that both liver and spleen of can respond to poly (I:C) challenge may be through promoting apoptosis of DNA-damaged cells, increasing the activity of immune-enhancing enzymes, and increasing energy supply based on DEGs annotation information.
CONCLUSIONS
Our results demonstrate the transcriptional responses of to poly (I:C)-stimulation, and these data provide the first resource on the genetic regulation mechanisms of against viruses. Furthermore, the present study can provide basic information for the prevention of viral diseases in artificial culture process.
Topics: Spleen; Poly I-C; Liver; Apoptosis; DNA Damage
PubMed: 37901224
DOI: 10.3389/fimmu.2023.1272393 -
Developmental and Comparative Immunology Dec 2023Camelids are economically and socially important in several parts of the world and might carry pathogens with epizootic or zoonotic potential. However, biological...
Camelids are economically and socially important in several parts of the world and might carry pathogens with epizootic or zoonotic potential. However, biological research in these species is limited due to lack of reagents. Here, we developed RT-qPCR assays to quantify a panel of camelid innate and adaptive immune response genes, which can be monitored in a single run. The assays were validated with PHA, PMA-ionomycin, and Poly I:C-stimulated PBMCs from alpaca, dromedary camel and llama, including normalization by multiple reference genes. Further, comparative gene expression analyses for the different camelid species were performed by a unique microfluidic qPCR assay. Compared to unstimulated controls, PHA and PMA-ionomycin stimulation elicited robust Th1 and Th2 responses in PBMCs from camelid species. Additional activation of type I and type III IFN signalling pathways was described exclusively in PHA-stimulated dromedary lymphocytes, in contrast to those from alpaca and llama. We also found that PolyI:C stimulation induced robust antiviral response genes in alpaca PBMCs. The proposed methodology should be useful for the measurement of immune responses to infection or vaccination in camelid species.
Topics: Animals; Cytokines; Camelids, New World; Camelus; Ionomycin; Microfluidics; RNA, Messenger
PubMed: 37717710
DOI: 10.1016/j.dci.2023.105061 -
Microbiology Spectrum Sep 2023The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of...
The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4 and CD8 T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections. IMPORTANCE The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.
PubMed: 37707204
DOI: 10.1128/spectrum.02192-23 -
Immunopharmacology and Immunotoxicology Dec 2023Toll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules....
OBJECTIVE
Toll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules. Ligand-triggered dimerization of TLR4 is essential for the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Saquinavir (SQV), an FDA-approved HIV protease inhibitor, has been shown to attenuate the activation of NF-κB induced by HMGB1 by blocking TLR4-MyD88 association in proteasome independent pathway. This study aims to define whether SQV is an HMGB1-specific and MyD88-dependent TLR4 signaling inhibitor and which precise signaling element of TLR4 is targeted by SQV.
MATERIALS AND METHODS
PMA differentiated human THP-1 macrophages or reconstituted HEK293 cells were pretreated with SQV before stimulated by different TLR agonists. TNF-α level was evaluated through ELISA assay. NF-κB activation was analyzed using NF-κB SEAP reporting system. The levels of MyD88/TRIF pathways-related factors were examined by immunoblot. TLR4 endocytosis was assessed by immunocytochemistry. TLR4 dimerization was determined using immunoprecipitation between different tagged TLR4 and an in silico molecular docking experiment was performed to explore the possible binding site of SQV on its target.
RESULTS
Our data showed that SQV suppresses both MyD88- and TRIF-dependent pathways in response to lipopolysaccharide (LPS), a critical sepsis inducer and TLR4 agonist, leading to downregulation of NF-κB and IRF3. SQV did not suppress MyD88-dependent pathway triggered by TLR1/2 agonist Pam3csk4. In the only TRIF-dependent pathway, SQV did not alleviate IRF3 phosphorylation induced by TLR3 agonist Poly(I:C). Furthermore, dimerization of TLR4 following LPS or HMGB1 stimulation was decreased by SQV.
CONCLUSION
We concluded that TLR4 receptor complex is one of the mammalian targets of SQV, and TLR4-mediated immune responses and consequent risk for uncontrolled inflammation could be modulated by FDA-approved drug SQV.
Topics: Animals; Humans; Toll-Like Receptor 4; Saquinavir; NF-kappa B; HIV Protease Inhibitors; HMGB1 Protein; Myeloid Differentiation Factor 88; Dimerization; Lipopolysaccharides; HEK293 Cells; Molecular Docking Simulation; Adaptor Proteins, Vesicular Transport; Mammals
PubMed: 37485845
DOI: 10.1080/08923973.2023.2239488 -
Extracellular adenosine triphosphate regulates inflammatory responses of periodontal ligament cells.Journal of Periodontology Mar 2024Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs)....
BACKGROUND
Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment.
METHODS
HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific PX purinoreceptor 7 (PX) inhibitors (brilliant blue G [BBG] and KN62), a specific PY purinoreceptor 1 (PY) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses.
RESULTS
LPS and poly I:C induced ATP release. A low concentration of eATP (50 µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500 µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10.
CONCLUSION
HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.
Topics: Humans; Periodontal Ligament; Adenosine Triphosphate; Lipopolysaccharides; Colforsin; Interleukin-10; Interleukin-4; Inflammation; Anti-Inflammatory Agents; Cells, Cultured; Poly I; Isoquinolines; Sulfonamides
PubMed: 37932872
DOI: 10.1002/JPER.23-0389 -
Developmental and Comparative Immunology Dec 2023In mammals, right open reading frame kinase 3 (RIOK3) is related with cancer development and immune regulation. To explore the role of teleost RIOK3 in the antiviral...
In mammals, right open reading frame kinase 3 (RIOK3) is related with cancer development and immune regulation. To explore the role of teleost RIOK3 in the antiviral innate immunity, the homolog of RIOK3 (bcRIOK3) from black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Sequence analysis revealed that bcRIOK3 is conserved in vertebrates. The transcription of bcRIOK3 varied in host cells in response to the stimulation of spring viremia of carp virus (SVCV), poly (I:C), and LPS. Immunoblotting (IB) and immunofluorescence (IF) assays identified bcRIOK3 as a cytoplasmic protein with a molecular weight of ∼60 kDa. It was interesting that bcRIOK3 knockdown led to the decreased basal mRNA levels of IFNa, IFNb and Viperin; however, triggered obviously higher mRNA levels of the above genes after viral infection and enhanced host resistance to SVCV. Like its mammalian counterpart, bcRIOK3 overexpression in EPC cells showed a significant inhibitory effect on black carp MDA5 (bcMDA5)-mediated transcription of interferon promoters and antiviral activity. Co-immunoprecipitation and immunofluorescent assays identified the association between bcRIOK3 and bcMDA5. Further analysis revealed that bcRIOK3 enhanced the K48-linked ubiquitination and proteasome-dependent degradation of bcMDA5, and it weakened the oligomerization of bcMDA5 under poly (I:C) stimulation. In summary, our data conclude that RIOK3 dampens MDA5-mediated IFN signaling by promoting its degradation in black carp, which provide new insights into the regulation of IFN signaling in teleost.
Topics: Animals; Humans; Carps; Rhabdoviridae; Reoviridae; Reoviridae Infections; Rhabdoviridae Infections; Antiviral Agents; Immunity, Innate; Poly I-C; RNA, Messenger; Fish Diseases; Fish Proteins; Mammals
PubMed: 37722630
DOI: 10.1016/j.dci.2023.105059 -
Veterinary Microbiology Sep 2023The type I interferon (IFN-I) is a critical component of the innate immune responses, and Coronaviruses (CoVs) from both the Alphacoronavirus and Betacoronavirus genera...
The type I interferon (IFN-I) is a critical component of the innate immune responses, and Coronaviruses (CoVs) from both the Alphacoronavirus and Betacoronavirus genera interfere with the IFN-I signaling pathway in various ways. Of the gammacoronaviruses that mainly infect birds, little is known about how infectious bronchitis virus (IBV), evades or interferes with the innate immune responses in avian hosts since few IBV strains have been adapted to grow in avian passage cells. Previously, we reported that a highly pathogenic IBV strain GD17/04 has adaptability in an avian cell line, providing a material basis for further study on the interaction mechanism. In the present work, we describe the suppression of IBV to IFN-I and the potential role of IBV-encoded nucleocapsid (N) protein. We show that IBV significantly inhibits the poly I: C-induced IFN-I production, accordingly the nuclear translocation of STAT1, and the expression of IFN-stimulated genes (ISGs). A detailed analysis revealed that N protein, acting as an IFN-I antagonist, significantly impedes the activation of the IFN-β promoter stimulated by MDA5 and LGP2 but does not counteract its activation by MAVS, TBK1, and IRF7. Further results showed that IBV N protein, verified to be an RNA-binding protein, interferes with MDA5 recognizing double-stranded RNA (dsRNA). Moreover, we found that the N protein targets LGP2, which is required in the chicken IFN-I signaling pathway. Taken together, this study provides a comprehensive analysis of the mechanism by which IBV evades avian innate immune responses.
Topics: Animals; Nucleocapsid Proteins; Infectious bronchitis virus; RNA, Double-Stranded; Signal Transduction; Interferon Type I
PubMed: 37307767
DOI: 10.1016/j.vetmic.2023.109798 -
Behavioural Brain Research Feb 2024Calorie restriction (CR) has been shown to extend the mean and maximum lifespan in both preclinical and clinical settings. We have previously demonstrated that CR...
Calorie restriction (CR) has been shown to extend the mean and maximum lifespan in both preclinical and clinical settings. We have previously demonstrated that CR attenuates lipopolysaccharide (LPS)-induced fever and sickness behavior. CR also leads to reductions in pro-inflammatory and increases in anti-inflammatory profiles. LPS is a bacterial mimetic; however, few studies have explored this phenomenon utilizing a viral mimetic, such as polyinosinic:polycytidylic acid (poly I:C). Dose-dependently, poly I:C induced an increase in core body temperature (T), with the largest dose (5000 µg/kg) resulting in a 1.62 °C ( ± 0.23 °C) T increase at 7 h post-injection in ad libitum mice and was associated with reduced home-cage locomotor activity. We then investigated the effect of 50% CR for 28 days to attenuate fever and sickness behavior induced by a poly I:C (5000 µg/kg) viral immune challenge. CR resulted in the partial attenuation of fever and sickness behavior measures post-poly I:C. The freely fed, control mice demonstrated a 2.02 °C ( ± 0.22 °C) increase in T at 7 h post-injection compared to the CR poly I:C group which demonstrated an increase in T of 0.94 °C ( ± 0.27 °C). Locomotor patterns post-injection were different, CR mice displayed a reduction in activity during the light phase, and the control group displayed a reduction during the dark phase. CR moderately attenuated the neuroinflammatory response with a reduction in microglial density in the ventromedial nucleus of the hypothalamus. The fever and sickness behavior attenuation seen after CR may be driven by similar anti-inflammatory processes as after LPS; however, further investigation is required.
Topics: Mice; Animals; Illness Behavior; Caloric Restriction; Lipopolysaccharides; Fever; Poly I-C; Anti-Inflammatory Agents
PubMed: 37838243
DOI: 10.1016/j.bbr.2023.114715