Poly (I:C)-induced maternal immune activation generates impairment of reversal learning performance in offspring.

Maternal immune activation (MIA) induces a variety of behavioral and brain abnormalities in offspring of rodent models, compatible with neurodevelopmental disorders, such as schizophrenia or autism. However, it remains controversial whether MIA impairs reversal learning, a basic expression of cognitive flexibility that seems to be altered in schizophrenia. In the present study, MIA was induced by administration of a single dose of polyriboinosinic-polyribocytidylic acid (Poly (I:C) (5 mg/kg i.p.)) or saline to mouse pregnant dams in gestational day (GD) 9.5. Immune activation was monitored through changes in weight and temperature. The offspring were evaluated when they reached adulthood (8 weeks) using a touchscreen-based system to investigate the effects of Poly (I:C) on discrimination and reversal learning performance. After an initial pre-training, mice were trained to discriminate between two different stimuli, of which only one was rewarded (acquisition phase). When the correct response reached above 80% values for two consecutive days, the images were reversed (reversal phase) to assess the adaptation capacity to a changing environment. Maternal Poly (I:C) treatment did not interfere with the learning process but induced deficits in reversal learning compared to control saline animals. Thus, the accuracy in the reversal phase was lower, and Poly (I:C) animals required more sessions to complete it, suggesting impairments in cognitive flexibility. This study advances the knowledge of how MIA affects behavior, especially cognitive domains that are impaired in schizophrenia. The findings support the validity of the Poly (I:C)-based MIA model as a tool to develop pharmacological treatments targeting cognitive deficits associated with neurodevelopmental disorders.

Inhibition of NKCC1 Ameliorates Anxiety and Autistic Behaviors Induced by Maternal Immune Activation in Mice.

Autism spectrum disorder (ASD) is thought to result from susceptibility genotypes and environmental risk factors. The offspring of women who experience pregnancy infection have an increased risk for autism. Maternal immune activation (MIA) in pregnant animals produces offspring with autistic behaviors, making MIA a useful model for autism. However, how MIA causes autistic behaviors in offspring is not fully understood. Here, we show that NKCC1 is critical for mediating autistic behaviors in MIA offspring. We confirmed that MIA induced by poly(I:C) infection during pregnancy leads to autistic behaviors in offspring. We further demonstrated that MIA offspring showed significant microglia activation, excessive dendritic spines, and narrow postsynaptic density (PSD) in their prefrontal cortex (PFC). Then, we discovered that these abnormalities may be caused by overexpression of NKCC1 in MIA offspring's PFCs. Finally, we ameliorated the autistic behaviors using PFC microinjection of NKCC1 inhibitor bumetanide (BTN) in MIA offspring. Our findings may shed new light on the pathological mechanisms for autism caused by pregnancy infection.

Maternal immune activation exerts long-term effects on activity and sleep in male offspring mice.

Exposure to infectious or non-infectious immune activation during early development is a serious risk factor for long-term behavioural dysfunctions. Mouse models of maternal immune activation (MIA) have increasingly been used to address neuronal and behavioural dysfunctions in response to prenatal infections. One commonly employed MIA model involves administering poly(I:C) (polyriboinosinic-polyribocytdilic acid), a synthetic analogue of double-stranded RNA, during gestation, which robustly induces an acute viral-like inflammatory response. Using electroencephalography (EEG) and infrared (IR) activity recordings, we explored alterations in sleep/wake, circadian and locomotor activity patterns on the adult male offspring of poly(I:C)-treated mothers. Our findings demonstrate that these offspring displayed reduced home cage activity during the (subjective) night under both light/dark or constant darkness conditions. In line with this finding, these mice exhibited an increase in non-rapid eye movement (NREM) sleep duration as well as an increase in sleep spindles density. Following sleep deprivation, poly(I:C)-exposed offspring extended NREM sleep duration and prolonged NREM sleep bouts during the dark phase as compared with non-exposed mice. Additionally, these mice exhibited a significant alteration in NREM sleep EEG spectral power under heightened sleep pressure. Together, our study highlights the lasting effects of infection and/or immune activation during pregnancy on circadian activity and sleep/wake patterns in the offspring.

Maternal selenium dietary supplementation alters sociability and reinforcement learning deficits induced by in utero exposure to maternal immune activation in mice.

Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.

Synaptic proteome perturbations after maternal immune activation: Identification of embryonic and adult hippocampal changes.

BACKGROUND: Maternal immune activation (MIA) triggers neurobiological changes in offspring, potentially reshaping the molecular synaptic landscape, with the hippocampus being particularly vulnerable. However, critical details regarding developmental timing of these changes and whether they differ between males and females remain unclear. METHODS: We induced MIA in C57BL/6J mice on gestational day nine using the viral mimetic poly(I:C) and performed mass spectrometry-based proteomic analyses on hippocampal synaptoneurosomes of embryonic (E18) and adult (20 ± 1 weeks) MIA offspring. RESULTS: In the embryonic synaptoneurosomes, MIA led to lipid, polysaccharide, and glycoprotein metabolism pathway disruptions. In the adult synaptic proteome, we observed a dynamic shift toward transmembrane trafficking, intracellular signalling cascades, including cell death and growth, and cytoskeletal organisation. In adults, many associated pathways overlapped between males and females. However, we found distinct sex-specific enrichment of dopaminergic and glutamatergic pathways. We identified 50 proteins altered by MIA in both embryonic and adult samples (28 with the same directionality), mainly involved in presynaptic structure and synaptic vesicle function. We probed human phenome-wide association study data in the cognitive and psychiatric domains, and 49 of the 50 genes encoding these proteins were significantly associated with the investigated phenotypes. CONCLUSIONS: Our data emphasise the dynamic effects of viral-like MIA on developing and mature hippocampi and provide novel targets for study following prenatal immune challenges. The 22 proteins that changed directionality from the embryonic to adult hippocampus, suggestive of compensatory over-adaptions, are particularly attractive for future investigations.

Knockdown of Tlr3 in dorsal striatum reduces ethanol consumption and acute functional tolerance in male mice.

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Adolescent intermittent ethanol (AIE) sensitized fever in male Sprague Dawley rats exposed to poly I:C in adulthood.

Fever plays an indispensable role in host defense processes and is used as a rapid index of infection severity. Unfortunately, there are also substantial individual differences in fever reactions with biological sex, immunological history, and other demographic variables contributing to adverse outcomes of infection. The present series of studies were designed to test the hypothesis that a history of adolescent alcohol misuse may be a latent experiential variable that determines fever severity using polyinosinic:polycytidylic acid (poly I:C), a synthetic form of double-stranded RNA that mimics a viral challenge. Adult male and female Sprague Dawley rats were injected with 0 (saline) or 4 mg/kg poly I:C to first establish sex differences in fever sensitivity in Experiment 1 using implanted radiotelemetry devices for remote tracking. In Experiments 2 and 3, adolescent males and females were exposed to either water or ethanol (0 or 4 g/kg intragastrically, 3 days on, 2 days off, ∼P30-P50, 4 cycles/12 exposures total). After a period of abstinence, adult rats (∼P80-96) were then challenged with saline or poly I:C, and fever induction and maintenance were examined across a prolonged time course of 8 h using implanted probes. In Experiments 4 and 5, adult male and female subjects with a prior history of adolescent water or adolescent intermittent ethanol (AIE) were given saline or poly I:C, with tissue collected for protein and gene expression analysis at 5 h post-injection. Initial sex differences in fever sensitivity were minimal in response to the 4 mg/kg dose of poly I:C in ethanol-naïve rats. AIE exposed males injected with poly I:C showed a sensitized fever response as well as enhanced TLR3, IκBα, and IL-1ß expression in the nucleus of the solitary tract. Other brain regions related to thermoregulation and peripheral organs such as spleen, liver, and blood showed generalized immune responses to poly I:C, with no differences evident between AIE and water-exposed males. In contrast, AIE did not affect responsiveness to poly I:C in females. Thus, the present findings suggest that adolescent binge drinking may produce sex-specific and long-lasting effects on fever reactivity to viral infection, with preliminary evidence suggesting that these effects may be due to centrally-mediated changes in fever regulation rather than peripheral immunological mechanisms.

Frequent low-impact exposure to THC during adolescence causes persistent sexually dimorphic alterations in the response to viral infection in mice.

Adolescent exposure to Δ9-tetrahydrocannabinol (THC) has enduring effects on energy metabolism and immune function. Prior work showed that daily administration of a low-impact dose of THC (5 mg/kg, intraperitoneal) during adolescence alters transcription in adult microglia and disrupts their response to bacterial endotoxin or social stress. To explore the lasting impact of adolescent THC exposure on the brain's reaction to viral infection, we administered THC (5 mg/kg, intraperitoneal) in male and female mice once daily on postnatal day (PND) 30-43. When the mice reached adulthood (PND 70), we challenged them with the viral mimic, polyinosinic acid:polycytidylic acid [Poly(I:C)], and assessed sickness behavior (motor activity, body temperature) and whole brain gene transcription. Poly(I:C) caused an elevation in body temperature which was lessened by prior THC exposure in female but not male mice. Adolescent THC exposure did not affect the locomotor response to Poly(I:C) in either sex. Transcriptomic analyses showed that Poly(I:C) produced a substantial upregulation of immune-related genes in the brain, which was decreased by THC in females. Additionally, the viral mimic caused a male-selective downregulation in transcription of genes involved in neurodevelopment and synaptic transmission, which was abrogated by adolescent THC treatment. The results indicate that Poly(I:C) produces complex transcriptional alterations in the mouse brain, which are sexually dimorphic and differentially affected by early-life THC exposure. In particular, adolescent THC dampens the brain's antiviral response to Poly(I:C) in female mice and prevents the transcriptional downregulation of neuron-related genes caused by the viral mimic in male mice.

Poly(I:C) induces anti-inflammatory response against secondary LPS challenge in zebrafish larvae.

Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages' number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

Involvement of mitochondrial fatty acid ß-oxidation in the antiviral innate immune response in head kidney macrophages of large yellow croaker (Larimichthys crocea).

As a vital pathway for cellular energy production, mitochondrial fatty acid ß-oxidation (FAO) is essential in regulating immune responses to bacterial pathogens and maintaining intracellular homeostasis in vertebrates. However, the specific role of FAO in antiviral innate immune response in macrophages remains insufficiently understood. In this study, virus infection simulated by poly(I:C) inhibited FAO, as indicated by the reduced expression of FAO-related genes and proteins in the head kidney of large yellow croaker, with similar results observed in poly(I:C)-stimulated macrophages. Then, inhibition of FAO by supplementary mildronate in vivo and etomoxir treatment in vitro revealed varying increases in the mRNA expression of antiviral innate immune response genes after stimulated by poly(I:C) in the head kidney and macrophages. Notably, etomoxir significantly facilitated the transcriptional up-regulation of the IFNh promoter by IRF3. Moreover, inhibiting FAO by knockdown of cpt1b promoted antiviral innate immune response triggered by poly(I:C) in macrophages. Conversely, activating FAO through overexpression of cpt1b or cpt2 significantly reduced the mRNA levels of antiviral response genes in macrophages stimulated by poly(I:C). Unlike etomoxir, cpt1b overexpression inhibited the transcriptional up-regulation of the IFNh promoter by IRF3. Furthermore, in vivo dietary palm oil feeding and in vitro exposure to palmitic acid inhibited the antiviral innate immune response triggered by poly(I:C) in the head kidney and macrophages, respectively. These effects were partly associated with FAO activation, as evidenced by etomoxir. In summary, this study elucidates FAO's critical role in regulating antiviral innate immune response in head kidney macrophages. These findings not only deepen insights into the interaction between metabolic remodeling and host immune responses, but also offer valuable guidance for developing nutritional strategies to improve antiviral immunity in aquaculture.

Short time-series expression transcriptome data reveal the gene expression patterns and potential biomarkers of blood infection with LPS and poly (I:C) in Mandarin fish (Siniperca chuatsi).

Blood transcriptomics has emerged as a vital tool for tracking the immune system and supporting disease diagnosis, prognosis, treatment, and research. The present study was conducted to analyze the gene expression profile and potential biomarker candidates using the whole blood of mandarin fish (Siniperca chuatsi) infected with LPS or poly (I:C) at 0 h, 3 h, 6 h, and 12 h. Our data suggest that 310 shared differentially expressed genes (DEGs) were identified among each comparison group after LPS infection, and 137 shared DEGs were identified after poly (I:C) infection. A total of 62 shared DEGs were differentially expressed in all compared groups after LPS or poly (I:C) infection. Pathways analysis for DEGs in all different compared groups showed that cytokine-cytokine receptor interaction was the most enrichment pathway. The expression levels of genes C-X-C chemokine receptor type 2-like (cxcr2), chemokine (C-C motif) receptor 9a (ccr9a), chemokine (C-C motif) receptor 9b (ccr9b), chemokine (C-X-C motif) receptor 4b (cxcr4b), and interleukin 10 receptor alpha (il10ra) were significantly different in all compared groups and most enriched in cytokine-cytokine receptor interaction pathway. The protein-protein interactions analysis among all shared DEGs showed that cxcr4 was the hub gene with the highest degree. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring mandarin fish diseases.

Transcriptomic and functional analysis of the antiviral response of mussels after a poly I:C stimulation.

The study of mussels (Mytilus galloprovincialis) has grown in importance in recent years due to their high economic value and resistance to pathogens. Because of the biological characteristics revealed by mussel genome sequencing, this species is a valuable research model. The high genomic variability and diversity, particularly in immune genes, may be responsible for their resistance to pathogens found in seawater and continuously filtered and internalized by them. These facts, combined with the lack of proven mussel susceptibility to viruses in comparison to other bivalves such as oysters, result in a lack of studies on mussel antiviral response. We used RNA-seq to examine the genomic response of mussel hemocytes after they were exposed to poly I:C, simulating immune cell contact with viral dsRNA. Apoptosis and the molecular axis IRFs/STING-IFI44/IRGC1 were identified as the two main pathways in charge of the response but we also found a modulation of lncRNAs. Finally, in order to obtain new information about the response of mussels to putative natural challenges, we used VHSV virus (Viral Hemorrhagic Septicemia Virus) to run some functional analysis and confirm poly I:C's activity as an immunomodulator in a VHSV waterborne stimulation. Both, poly I:C as well as an injury stimulus (filtered sea water injection) accelerated the viral clearance by hemocytes and altered the expression of several immune genes, including IL-17, IRF1 and viperin.

Establishment and characterization of a golden pompano (Trachinotus blochii) fin cell line for applications in marine fish pathogen immunology.

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1ß, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco).

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequences alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. In summary, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

Members of the TRAF gene family in Octopus sinensis and their response to PGN, poly I:C, and Vibrio parahaemolyticus.

Octopus sinensis, the species of Cephalopoda, is known as the highest Mollusca and is an economic and new aquaculture species in the coastal waters of southern China. The immune system has been well documented to have a function of resisting the invasion of pathogens in the external environment among mollusca species. As a kind of signaling molecule in the innate immune system, tumor necrosis factor (TNF) receptor-associated factor (TRAF) plays significant roles in TNF receptor (TNFR)/interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) signaling pathways. Until now, seven TRAF members (TRAF1-7) have been discovered, and they have been reported to participate in regulating signal pathways mediated by pattern recognition receptors and play important roles in the innate immune response of the hosts. In this study, five TRAF genes of O. sinensis (OsTRAF2, OsTRAF3, OsTRAF4, OsTRAF6, and OsTRAF7) were identified, whose full length of the open reading frame is 1473 bp, 1629 bp, 1431 bp, 1353 bp and 2121 bp respectively, encoding 490, 542, 476, 450 and 706 amino acids, respectively. Bioinformatics analysis showed that each OsTRAF has different chromosome locations. In addition to seven consecutive WD40 domains on the C-terminal of OsTRAF7 protein, the C-terminal of OsTRAF proteins all contain a conserved TRAF domain, namely the MATH domain. Phylogenetic analysis showed that OsTRAF proteins were clustered together with TRAF proteins of bivalves. Moreover, TRAF1 and TRAF2, TRAF3 and TRAF5 were clustered together in a large clade, respectively, revealing they have a close genetic relationship. The results of quantitative Real-time PCR showed that OsTRAF genes were highly expressed in the gill, hepatopancreas and white body. After stimulation with PGN, poly I:C and V. parahaemolyticus, the expression levels of OsTRAF genes were up-regulated in the gill, hepatopancreas and white body at different time points. These results indicated that OsTRAF genes play an important role in the antibacterial and antiviral immune response of O. sinensis.

Efficacy of IFN-γ, sCD40L, and Poly(I:C) Treated Bone Marrow-Derived Macrophages in Murine Mammary Carcinoma.

INTRODUCTION: Here, we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites could impact disease progression. METHODS: BMDM treated with IFN-γ, sCD40L, poly(I:C), and a combination of the three were assessed. RESULTS: Treatment with sCD40L had no significant impact on the BMDM. Treating BMDM with IFN-γ impacted IL-1ß, MHC Class II, and CD80 expression. While poly(I:C) treatment had a greater impact on the BMDM than IFN-γ when assessed by the in vitro assays, the BMDM treated with poly (I:C) had mixed results in vivo where they decreased growth of the EMT6 tumor, did not impact growth of the 168 tumor, and enhanced growth of the 4T1 tumor. The combination of poly(I:C), IFN-γ, and sCD40L had the greatest impact on the BMDM in vitro and in vivo. Treatment with all three agonists resulted in increased IL-1ß, TNF-α, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, nitrite production, and MHC Class II and CD80 expression, and significantly impacted growth of the EMT6 and 168 murine mammary carcinoma models. DISCUSSION: Collectively, these data show that treating BMDM with poly(I:C), IFN-γ, and sCD40L generates BMDM with more consistent anti-tumor activity than BMDM generated with the individual agonists.

Late prenatal immune activation in mice induces transgenerational effects via the maternal and paternal lineages.

Prenatal exposure to infectious or noninfectious immune activation is an environmental risk factor for neurodevelopmental disorders and mental illnesses. Recent research using animal models suggests that maternal immune activation (MIA) during early to middle stages of pregnancy can induce transgenerational effects on brain and behavior, likely via inducing stable epigenetic modifications across generations. Using a mouse model of viral-like MIA, which is based on gestational treatment with poly(I:C), the present study explored whether transgenerational effects can also emerge when MIA occurs in late pregnancy. Our findings demonstrate that the direct descendants born to poly(I:C)-treated mothers display deficits in temporal order memory, which are similarly present in second- and third-generation offspring. These transgenerational effects were mediated via both the maternal and paternal lineages and were accompanied by transient changes in maternal care. In addition to the cognitive effects, late prenatal immune activation induced generation-spanning effects on the prefrontal expression of gamma-aminobutyric acid (GABA)ergic genes, including parvalbumin and distinct alpha-subunits of the GABAA receptor. Together, our results suggest that MIA in late pregnancy has the potential to affect cognitive functions and prefrontal gene expression patterns in multiple generations, highlighting its role in shaping disease risk across generations.

Effects of neuropeptide Y on the immune-protection and intestinal tract of juvenile Micropterus salmoides.

Neuropeptide Y is known to be directly or indirectly involved in immune regulation. The immune effects of NPY include immune cell transport, helper T cell differentiation, cytokine secretion, staining and killer cell activity, phagocytosis and production of reactive oxygen species. In this study, we investigated the immunoprotective effect of synthetic NPY on largemouth bass larvae. For the first time, the dose and time effects of NPY injection on largemouth bass was explored, and then Poly I:C and LPS infection was carried out in juvenile largemouth bass, respectively, after the injection of NPY. The results showed that NPY could reduce the inflammatory response by inhibiting the expression of il-1ß, tgf-ß, ifn-γ and other immune factors in head kidney, spleen and brain, and alleviate the immune stress caused by strong inflammatory response in the early stage of infection. Meanwhile, NPY injection ameliorated the intestinal tissue damage caused by infection. This study provides a new way to protect juvenile fish and improve its innate immunity.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.