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More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

Immunotherapy is currently under intensive investigation as a potential breakthrough treatment option for glioblastoma. Given the anatomical and immunological complexities surrounding glioblastoma, lymphocytes that infiltrate the brain to develop durable immunity with memory will be key. Polyinosinic:polycytidylic acid, or poly(I:C), and its derivative poly-ICLC could serve as a priming or boosting therapy to unleash lymphocytes and other factors in the (immuno)therapeutic armory against glioblastoma. Here, we present a systematic review on the effects and efficacy of poly(I:C)/poly-ICLC for glioblastoma treatment, ranging from preclinical work on cellular and murine glioblastoma models to reported and ongoing clinical studies. MEDLINE was searched until 15 May 2021 to identify preclinical (glioblastoma cells, murine models) and clinical studies that investigated poly(I:C) or poly-ICLC in glioblastoma. A systematic review approach was conducted according to PRISMA guidelines.ClinicalTrials.govwas queried for ongoing clinical studies. Direct pro-tumorigenic effects of poly(I:C) on glioblastoma cells have not been described. On the contrary, poly(I:C) changes the immunological profile of glioblastoma cells and can also kill them directly. In murine glioblastoma models, poly(I:C) has shown therapeutic relevance as an adjuvant therapy to several treatment modalities, including vaccination and immune checkpoint blockade. Clinically, mostly as an adjuvant to dendritic cell or peptide vaccines, poly-ICLC has been demonstrated to be safe and capable of eliciting immunological activity to boost therapeutic responses. Poly-ICLC could be a valuable tool to enhance immunotherapeutic approaches for glioblastoma. We conclude by proposing several promising combination strategies that might advance glioblastoma immunotherapy and discuss key pre-clinical aspects to improve clinical translation.

Maternal immune activation (MIA) is mediated by activation of inflammatory pathways resulting in increased levels of cytokines and chemokines that cross the placental and blood-brain barriers altering fetal neural development. Maternal viral infection is one of the most well-known causes for immune activation in pregnant women. MIA and immune abnormalities are key players in the etiology of developmental conditions such as autism, schizophrenia, ADHD, and depression. Experimental evidence implicating MIA in with different effects in the offspring is complex. For decades, scientists have relied on either MIA models or human epidemiological data or a combination of both. MIA models are generated using infection/pathogenic agents to induce an immunological reaction in rodents and monitor the effects. Human epidemiological studies investigate a link between maternal infection and/or high levels of cytokines in pregnant mothers and the likelihood of developing conditions. In this review, we discuss the importance of understanding the relationship between virus-mediated MIA and neurodevelopmental conditions, focusing on autism and schizophrenia. We further discuss the different methods of studying MIA and their limitations and focus on the different factors contributing to MIA heterogeneity.

Use of nanoparticles as drug carrier vectors has great potential to circumvent the limitations associated with chemotherapy, including drug resistance and destructive side effects. For this purpose, magnetic generation 4 dendrimeric nanoparticles were prepared to carry chemotherapeutic agent doxorubicin (G 4-DOX) and immune modulator polyinosinic:polycytidylic acid [Poly(I:C)]. As previously reported, DOX and Poly(I:C) was loaded onto G 4 nanoparticles (PIC-G 4-DOX). Cellular internalization study using confocal microscopy demonstrated high levels of cellular internalization of PIC-G 4-DOX nanoparticles by MCF-7 cells. This resulted in higher efficacy of PIC-G 4-DOX nanoparticles in killing MCF-7 breast cancer cells. Alteration in the expression levels of selected genes was determined by RT-qPCR analyses. Proapoptotic NOXA, PUMA, and BAX genes were upregulated, and SURVIVIN, APOLLON, and BCL-2 genes were downregulated, indicating the cell-killing effectiveness of PIC-G 4-DOX nanoparticles. Gene expression analysis provided some insights into the possible molecular mechanisms on cytotoxicity of DOX and Poly(I:C) delivered through G 4 magnetic nanoparticles. The results demonstrated that PIC-G 4-DOX can be useful for targeted delivery affecting apoptotic pathways, resulting in an advanced degree of cancer-cell-killing. They are promising for targeting cancer-cells because of their stability, biocompatibility, higher internalization, and toxicity.

Poly(I:C) is a promising adjuvant for cancer treatment vaccines to enhance the host anti‐tumour immune response. However, the roles of poly(I:C) in the cervical cancer microenvironment and local immune reactions are not well understood. In this study, we investigated the roles of poly(I:C) in the cervical cancer. We analysed the cytokine transcription and secretion of cervical cancer cell lines and THP‐1–derived macrophages after poly(I:C) treatment, respectively. These results revealed that IL‐6 was significantly up‐regulated, and this up‐regulation was partly dose dependent. poly(I:C)‐stimulated supernatant of cervical cancer cells promoted M1‐type cytokine IL‐1β and IL‐6 expression of THP‐1–derived macrophages, but inhibited the expression of M2‐type cytokine, IL‐10 and CCL22. The recruitment of THP‐1–derived macrophages by poly(I:C)‐stimulated cervical cancer cell supernatant was also enhanced. Inhibition of IL‐6 expression in cervical cancer cells by siRNA transfection almost completely reversed the effects of poly(I:C) treatment. Finally, we found that phosphorylation of the NF‐κB signalling pathway in cervical cancer cells occurred quickly after poly(I:C) treatment. Moreover, the NF‐κB signalling pathway inhibitor PDTC significantly inhibited poly(I:C)‐induced IL‐6 expression. Taken together, these results suggest that poly(I:C) might regulate the effects of cervical cancer cells on tumour‐infiltrated macrophages, and subsequently promote a pro‐inflammatory tumour microenvironment.

Background Proper blocking of toll-like receptor (TLR) activation during disease progression has been reported to have inhibitory effect on the pathogenesis of rheumatoid arthritis (RA). We tested whether the TLR4 inhibitor TAK-242 had potential as a remedy for rheumatoid arthritis. Methods The therapeutic effect of TAK-242 was tested in vitro using the human rheumatoid fibroblast-like synoviocyte (FLS) line MH7A or primary human FLS and in an adjuvant-induced arthritis (AIA) rat model. Results TAK-242 dose dependently inhibited the increased expression of IL-6, IL-8, MMP-1, and VEGF in LPS-stimulated MH7A cells. It also inhibited the expression of IL-6 and IL-8 in poly(I:C), TLR3 activator-stimulated primary FLS, but not in IL-1β-stimulated primary FLS. These findings suggest that TAK-242 blocks a specific signaling pathway to some degree. Further, TAK-242 slightly inhibited mobilization of NF-κB into nuclei. In the AIA rat model, TAK-242 significantly reversed the body weight and paw thickness of AIA rats to the normal state at a dose of 5 mg/kg, but not at 3 mg/kg, and reduced the increased serum level of IL-6 and VEGF in AIA rats. It also significantly ameliorated inflammatory symptoms of joint tissues at day 21 of treatment, according to histology and RT-PCR. Conclusions Based on the drug repositioning concept, TAK-242, which is used for the treatment of TLR4-mediated inflammatory diseases, shows potential for cost-effective development as a remedy for rheumatoid arthritis or to control the progression of RA.

Background Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. Methods Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. Results Pre-treatment with 200 μg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3−) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. Conclusions Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis. Open-Access-Publikationsfonds 2020

Curcumin is widely used in dietary supplements due to its beneficial effects on inflammatory and immune pathways. To better understand the action of curcumin during viral infection, its effect on stimulation of RAW264.7 macrophages with polyinosinic-polycytidylic acid (poly(I:C)) was evaluated by deep sequencing transcriptome, followed by RT-qPCR, western blotting and ELISA validations. The transcriptomics results showed that 157 differentially expressed genes are in association with the activity of curcumin on the cellular response to poly(I:C) stimulation. Gene Ontology and KEGG enrichment analysis uncovered the involvement of TLRs, in particular TLR3, in curcumin’s effect on poly(I:C)-induced immune and inflammatory responses. RT-qPCR results showed that curcumin (2.5, 5, 10 μM) significantly down-regulated the mRNA expression of Tlr3, Irf-7, Ifnb1, Il-6, Tnfα, and Il1bf in a dose-dependent manner. Significant down-regulation of curcumin (5 M) on protein or phosphorylated protein expressions of TLR3 (p

UV-irradiation induces the secretion of double-stranded RNA (dsRNA) derived from damaged noncoding RNAs in keratinocytes, which enhance the expression of matrix metalloproteinases (MMP) in non-irradiated dermal fibroblasts, leading to dysregulation of extracellular matrix homeostasis. However, the signaling pathway responsible for dsRNA-induced MMP expression has not been fully understood. Transglutaminase 2 (TG2) is an enzyme that modifies substrate proteins by incorporating polyamine or crosslinking of proteins, thereby regulating their functions. In this study, we showed that TG2 mediates dsRNA-induced MMP-1 expression through NF-κB activation. Treatment of poly(I:C), a synthetic dsRNA analogue binding to toll-like receptor 3 (TLR3), generates ROS, which in turn activates TG2 in dermal fibroblast. Subsequently, TG2 activity enhances translocation of p65 into the nucleus, where it augments transcription of MMP. We confirmed these results by assessing the level of MMP expression in Tlr3−/−, TG2-knockdowned and Tgm2−/− dermal fibroblasts after poly(I:C)-treatment. Moreover, treatment with quercetin showed dose-dependent suppression of poly(I:C)-induced MMP expression. Furthermore, ex vivo cultured skin from Tgm2−/− mice exhibited a significantly reduced level of MMP mRNA compared with those from wild-type mice. Our results indicate that TG2 is a critical regulator in dsRNA-induced MMP expression, providing a new target and molecular basis for antioxidant therapy in preventing collagen degradation.

Zika virus (ZIKV) is an emerging teratogenic arbovirus that persists in semen and is sexually transmitted. We previously demonstrated that ZIKV infects the human testis and persists in testicular germ cells (TGCs) for several months after patients’ recovery. To decipher the mechanisms underlying prolonged ZIKV replication in TGCs, we compared the innate immune response of human testis explants and isolated TGCs to ZIKV and to Poly(I:C), a viral RNA analog. Our results demonstrate the weak innate responses of human testis to both ZIKV and Poly(I:C) as compared with other tissues or species. TGCs failed to up-regulate antiviral effectors and type I IFN upon ZIKV or Poly(I:C) stimulation, which might be due to a tight control of PRR signaling, as evidenced by the absence of activation of the downstream effector IRF3 and elevated expression of repressors. Importantly, exogenous IFNβ boosted the innate immunity of TGCs and inhibited ZIKV replication in the testis ex vivo, raising hopes for the prevention of ZIKV infection and persistence in this organ.

Cervical cancer is one of the most common malignancies in women, with a poor survival rate. Thus, there is a need to define effective combination strategies to improve therapy. In this study, we report that dsRNA poly(I:C) up-regulated the expression of IFNβ and apoptosis-associated genes in cervical cancer cells, activating both intrinsic and extrinsic apoptotic pathways, and eventually inducing cell death. Similarly, proteasome inhibitors also effectively induced cervical cancer cell apoptosis, probably through prevention of p53 degradation, inhibiting NF-κB signal activation and decreasing BCL-2 expression. Importantly, the combination of poly(I:C) with proteasome inhibitors enhanced caspase-8 and caspase-9 activation, and synergistically induced cervical cancer cell apoptosis. Both activated p38 signals and increased ROS levels, and their combination extended these effects. Collectively, we show that the activation of multiple pro-apoptotic pathways by poly(I:C) and proteasome inhibitors underpin a synergistic effect on inducing cervical cancer cell death, suggesting a potential therapeutic combination with clinical relevance.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1β, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.

Toll-like receptor 3 (TLR3) is a sensor of endogenous cell necrosis during the process of acute inflammation. Interleukin (IL)-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine and can negatively regulate the pathogenesis of inflammation. However, whether and how activation of TLR3 can regulate IL-1Ra expression has not been clarified. Here, we show that poly(I:C) induces IL-1Ra expression in primarily cultured human fibroblast-like synoviocytes and other types of cells. Induction of IL-1Ra by poly(I:C) was dependent on TLR3, but was independent of melanoma differentiation-­associated protein 5 or retinoic acid-inducible gene I. Interferon regulatory factor 3 (IRF3) directly binds to the IL-1Ra promoter and promotes IL-1Ra expression in response to poly(I:C) stimulation. Induction of IL-1Ra by poly(I:C) was abolished by the inhibition of the NF-κB signaling, attenuated by the inhibition of the PI3K-Akt signaling, enhanced by inhibition of the ERK1/2 or MSK1/2 activation, but was independent of the p38 MAPK signaling. Treatment with poly(I:C) or Sendai virus elevated the levels of serum IL-1Ra in wild-type, but not in TLR3–/– or IRF3–/– mice. Our findings may provide new insights into the intrinsic anti-inflammatory function of TLR3 and double-stranded RNA-induced IL-Ra expression by TLR3 and its regulation.

Poly(I:C) is a kind of chemosynthetic double-stranded RNA (dsRNA) analogue which could act as TLR3 agonist and induce IFN production. It is widely applied in anti-virus treatment and immunoregulation, as well as vaccine adjuvant in farmed animals. However, whether poly(I:C) could activate innate immune response to defense against bacterial infection remains unclear. In this study, we established a feeding trial model with different dose of poly(I:C) in turbot larvae, then challenged with Edwardsiella piscicida after 3–7 weeks resting period. The results show that feeding turbot with poly(I:C) exhibited a stronger inflammatory response and antioxidant stress ability, and significantly elevated the survival rate within the decreased bacterial loads. Importantly, the bacterial infection-induced white feces in hindgut of turbot were significantly alleviated after poly(I:C) feeding, and this administration induced protection could last for about 7 weeks. Taken together, these findings indicate that feeding turbot with poly(I:C) could enhance a long-term intestinal mucosal immunity in response to bacterial infection, suggesting that poly(I:C) might be a promising immunostimulant in aquaculture.

The respiratory tract is constantly at risk of invasion by microorganisms such as bacteria, viruses, and fungi. In particular, the mucosal epithelium of the nasal cavity and paranasal sinuses is at the very forefront of the battles between the host and the invading pathogens. Recent studies have revealed that the epithelium not only constitutes a physical barrier but also takes an essential role in the activation of the immune system. One of the mechanisms equipped in the epithelium to fight against microorganisms is the Toll-like receptor (TLR) response. TLRs recognize common structural components of microorganisms and activate the innate immune system, resulting in the production of a plethora of cytokines and chemokines in the response against microbes. As the epithelia-derived cytokines are deeply involved in the pathogenesis of inflammatory conditions in the nasal cavity and paranasal sinuses, such as chronic rhinosinusitis (CRS) and allergic rhinitis (AR), the molecules involved in the TLR response may be utilized as therapeutic targets for these diseases. There are several differences in the TLR response between nasal and bronchial epithelial cells, and knowledge of the TLR signals in the upper airway is sparse compared to that in the lower airway. In this review, we provide recent evidence on TLR signaling in the upper airway, focusing on the expression, regulation, and responsiveness of TLRs in human nasal epithelial cells (HNECs). We also discuss how TLRs in the epithelium are involved in the pathogenesis of, and possible therapeutic targeting, for CRS and AR.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Toll-like receptor (TLR) is a cluster of type I transmembrane proteins that plays a role in innate immunity. Based on the marbled rockfish (Sebastiscus marmoratus) genome database, this study used bioinformatics methods to identify and analyze its TLR gene family members. The results showed that there were 11 TLR gene family members in Sebastiscus marmoratus (SmaTLR), which could be divided into five different subfamilies. The number of amino acids encoded by the Smatlr genes ranged from 637 to 1206. The physicochemical properties of the encoded proteins of different members were also computed. The results of protein structure prediction, phylogenetic relation, and motif analysis showed that the structure and function of the SmaTLRs were relatively conserved. Quantitative Real-Time PCR (qRT-PCR) analysis revealed the expression patterns of SmaTLRs in the gill, liver, spleen, head kidney, kidney, and intestine. SmaTLRs were widely detected in the tested tissues, and they tended to be expressed higher in immune-related tissues. After polyriboinosinic polyribocytidylic acid (poly(I:C)) challenge, SmaTLR14, SmaTLR3, SmaTLR5S, SmaTLR7, and SmaTLR22 were significantly upregulated in the spleen or liver. The results of this study will help to understand the status of TLR gene family members of marbled rockfish and provide a basis for further study of the functional analysis of this gene family.

BackgroundThe mechanisms by which moderate tidal volume ventilation (MTV) exacerbates preexisting lung injury are unclear. We hypothesized that systemic endotoxemia via the gut-lung axis would lead to non-canonical and canonical inflammasome activation and pyroptosis in a two-hit model involving polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA and MTV and that this would associate with acute lung injury (ALI).MethodsAnesthetized mice were administered Poly(I:C) intratracheally and then 6 h later, they were mechanically ventilated for 4 h with otherwise non-injurious MTV (10ml/kg). Changes in intestinal and alveolar capillary permeability were measured. Further documentation of ALI was assessed by evans blue albumin permeability, protein and IL-1 family concentration in bronchoalveolar lavage fluid (BALF) or plasma, and histopathology in cohorts of wildtype (WT), whole body genetically ablated caspase-11 (caspase-11-/-), caspase-1/caspase-11 double knockout (caspase-1/11-/-), gasdermin D (GSDMD)-/-, nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3)-/- and advanced glycosylation end product-specific receptor (RAGE) -/- mice.ResultsNon-injurious MTV exacerbated the mild lung injury associated with Poly(I:C) administration. This included the disruption of alveolar-capillary barrier and increased levels of interleukin (IL)-6, high mobility group proteins 1 (HMGB-1), IL-1β in BALF and IL-18 in plasma. Combined (Poly(I:C)-MTV) injury was associated with increase in gastrointestinal permeability and endotoxin in plasma and BALF. Poly(I:C)-MTV injury was sensitive to caspase-11 deletion with no further contribution of caspase-1 except for maturation and release of IL-18 (that itself was sensitive to deletion of NLRP3). Combined injury led to large increases in caspase-1 and caspase-11. Genetic ablation of GSDMD attenuated alveolar-capillary disruption and release of cytokines in combined injury model.ConclusionsThe previously noted exacerbation of mild Poly(I:C)-induced ALI by otherwise non-injurious MTV is associated with an increase in gut permeability resulting in systemic endotoxemia. The gut-lung axis resulted in activation of pulmonary non-canonical (cytosolic mediated caspase-11 activation) and canonical (caspase-1) inflammasome (NLRP3) mediated ALI in this two-hit model resulting in GSDMD sensitive alveolar capillary barrier disruption, pyroptosis (alveolar macrophages) and cytokine maturation and release (IL-1β; IL-18). Pharmacologic strategies aimed at disrupting communication between gut and lung, inhibition of inflammasomes or GSDMD in pyroptosis may be useful in ALI.

Background Postviral olfactory dysfunction (PVOD) following a viral upper respiratory tract infection (URI) is one of the most common causes of olfactory disorders, often lasting for over a year. To date, the molecular pathology of PVOD has not been elucidated. Methods A murine model of Toll-like receptor 3 (TLR3)-mediated upper respiratory tract inflammation was used to investigate the impact of URIs on the olfactory system. Inflammation was induced via the intranasal administration of polyinosinic–polycytidylic acid (poly(I:C), a TLR3 ligand) to the right nostril for 3 days. Peripheral olfactory sensory neurons (OSNs), immune cells in the olfactory mucosa, and glial cells in the olfactory bulb (OB) were analyzed histologically. Proinflammatory cytokines in the nasal tissue and OB were evaluated using the quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Results In the treated mice, OSNs were markedly reduced in the olfactory mucosa, and T cell and neutrophil infiltration therein was observed 1 day after the end of poly(I:C) administration. Moreover, there was a considerable increase in microglial cells and slight increase in activated astrocytes in the OB. In addition, qPCR and ELISA revealed the elevated expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma both in the OB and nasal tissue. Conclusions Taken together, the decreased peripheral OSNs, OB microgliosis, and elevated proinflammatory cytokines suggest that immunological changes in the OB may be involved in the pathogenesis of PVOD.

Purpose: Retinoic acid-inducible gene-I （RIG-I） is a member of cytoplasmic viral sensors which plays an important role in inflammation of biliary epithelial cells（ BECs）. The aim of this study is to examine if RIG-I and C-X-C motif chemokine 10（ CXCL10） are involved in the etiology of human biliary atresia（ BA）. Methods: Immunohistochemical study was performed on surgically resected tissues obtained（ June 1994 to March 2016）from 30 infants with BA and non-inflamed hepatic tissues from 7 infants with hepatoblastoma. A semiquantitative scoring system was designed to evaluate the staining with an antibodies to the RIG-I and CXCL10. The expression of RIG-I and CXCL10 in HuCCT1 cholangiocarcinoma cell line were studied by western blotting, ELISA and RT-PCR analyses. Results: Intense immunoreactivity for RIG-I and CXCL10 was detected in BECs in tissues resected from BA patients.The expression of RIG-I and CXCL10 in the hilar tissue was significantly stronger than in the hepatic tissue.Transfection of HuCCT1 cells with poly（I:C）, a synthetic analog of viral dsRNA, induced the expression of RIG-I, and knockdown of RIG-I inhibited the induction of CXCL10 in HuCCT1 cells transfected with poly（I:C）. Conclusion: These results suggest that RIG-I-CXCL10 cascade may be involved in the etiology of human BA.

The aims of our study are to: (i) investigate the ability of nicotine to modulate the expression level of inflammatory cytokines in A549 cells infected with SARS-CoV-2; (ii) elucidate the ultrastructural features caused by the combination nicotine+SARS-CoV-2; and (iii) demonstrate the mechanism of action. In this study, A549 cells pretreated with nicotine were either exposed to LPS or poly(I:C), or infected with SARS-CoV-2. Treated and untreated cells were analyzed for cytokine production, cytotoxicity, and ultrastructural modifications. Vero E6 cells were used as a positive reference. Cells pretreated with nicotine showed a decrease of IL6 and TNFα in A549 cells induced by LPS or poly(I:C). In contrast, cells exposed to SARS-CoV-2 showed a high increase of IL6, IL8, IL10 and TNFα, high cytopathic effects that were dose- and time-dependent, and profound ultrastructural modifications. These modifications were characterized by membrane ruptures and fragmentation, the swelling of cytosol and mitochondria, the release of cytoplasmic content in extracellular spaces (including osmiophilic granules), the fragmentation of endoplasmic reticulum, and chromatin disorganization. Nicotine increased SARS-CoV-2 cytopathic effects, elevating the levels of inflammatory cytokines, and inducing severe cellular damage, with features resembling pyroptosis and necroptosis. The protective role of nicotine in COVID-19 is definitively ruled out.

Exosomes are small membrane vesicles that contain proteins and nucleic acids derived from secretory cells and mediate intracellular communication. Immune cell-derived exosomes regulate immune responses and gene expression of recipient cells. Macrophages recognize viral dsRNA via Toll-like receptor 3, thereby inducing the activation of transcription factors such as interferon regulatory factor 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In this study, we aimed to identify the immunomodulatory functions of exosomes derived from chicken macrophages (HD11) stimulated with polyinosinic-polycytidylic acid (poly[I:C]); exosomes were then delivered into HD11 cells and CU91 chicken T cells. Exosomes purified from poly(I:C)-activated macrophages stimulated the expression of type I interferons, proinflammatory cytokines, anti-inflammatory cytokines, and chemokines in HD11 and CU91 cells. Moreover, poly(I:C)-stimulated exosomes induced the NF-κB signaling pathway by phosphorylating TAK1 and NF-κB1. Therefore, we suggest that after the activation of Toll-like receptor 3 ligands following infection with dsRNA virus, chicken macrophages regulate the immune response of naive macrophages and T cells through the NF-κB signaling pathway. Furthermore, poly(I:C)-activated exosomes can be potentially utilized as immunostimulators.

Mesenchymal stem cells (MSCs) help fight infection by promoting direct bacterial killing or indirectly by modulating the acute phase response, thereby decreasing tissue injury. Recent evidence suggests that extracellular vesicles (EVs) released from MSCs retain antimicrobial characteristics that may be enhanced by pretreatment of parent MSCs with the toll-like receptor 3 (TLR3) agonist poly(I:C). Our aim was to determine whether poly(I:C) priming can modify EV content of miRNAs and/or proteins to gain insight into the molecular mechanisms of their enhanced antimicrobial function. Human bone marrow-derived MSCs were cultured with or without 1 μg/ml poly(I:C) for 1 h and then conditioned media was collected after 64 h of culture in EV-depleted media. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. Poly(I:C) priming resulted in 49 upregulated EV proteins, with 21 known to be important in host defense and innate immunity. In contrast, EV miRNA content was not significantly altered. Functional annotation clustering analysis revealed enrichment in biological processes and pathways including negative regulation of endopeptidase activity, acute phase, complement and coagulation cascades, innate immunity, immune response, and Staphylococcus aureus infection. Several antimicrobial peptides identified in EVs remained unaltered by poly(I:C) priming, including dermcidin, lactoferrin, lipocalin 1, lysozyme C, neutrophil defensin 1, S100A7 (psoriasin), S100A8/A9 (calprotectin), and histone H4. Although TLR3 activation of MSCs improves the proteomic profile of EVs, further investigation is needed to determine the relative importance of particular functional EV proteins and their activated signaling pathways following EV interaction with immune cells.

Evidences have suggested that Sjogren's syndrome (SS) is associated with viral infection. The aim of this study was to investigate the involvement of respiratory viral poly(I:C) in the pathogenesis of SS and potential mechanisms using a SS-like NOD/ShiLtJ (NOD) mouse model. 5-week female NOD mice were intratracheally administered poly(I:C) every other day for 5 times to mimic viral infection. Pilocarpine induced saliva secretion was determined every 8 days. Submandibular glands (SMG) and lungs were harvested for the detection of pathological changes. We found that intratracheal administration of poly(I:C) significantly advanced and enhanced the reduction of saliva flow rate in NOD mice. Furthermore, poly(I:C) treatment aggravated the histopathological lesions and inflammatory cells infiltration in SMG. Accompanied by elevated expression of IFN cytokines and IL-33, Th1 activation was enhanced in SMG of poly(I:C)-treated NOD mice, but Th17 cells activation was unchanged among the groups. In addition, intratracheal poly(I:C) exposure promoted the expression of IL-33 and increased T cells proportion in the lung, which were consistent with the change in SMG. Therefore, intratracheal poly(I:C) exposure aggravated the immunological and function disorder of SMG in NOD mice.

The combined effects on pig behavior of maternal immune challenge during gestation followed by a second immune challenge later in life have not been studied. Porcine reproductive and respiratory syndrome virus (PRRSV) infection during gestation can elicit maternal immune activation (MIA) yet the interactions with the offspring response to a second immune challenge after birth remains unexplored. Knowledge on the response to viral challenges in rodents has been gained through the use of the viral mimetic polyinosinic-polycytidylic acid (Poly(I:C)), yet the effects of this immune stimulant on pig behavior have not been assessed. This study advances the understanding of the combined effect of MIA and a second immune challenge later in life on female and male pig behavior. Three complementary experiments enabled the development of an effective Poly(I:C) challenge in pigs, and testing the interaction between PRRSV-elicited MIA, Poly(I:C) challenge at 60 days of age, and sex on behaviors. Individual-level observations on sickness, locomotor, and social behaviors were measured 1–3 h after Poly(I:C) challenge. Vomiting, panting, lethargy, walking, laying, playing, and touching behaviors were analyzed using generalized linear mixed effect models. Results indicated that a Poly(I:C) dose of 1 mg/kg within 1 h after injection increased the incidence of laying and sickness behavior. The Poly(I:C) challenge decreased the incidence of locomotor behaviors and activity levels. Pigs exposed to MIA had lower rates of social behaviors such as playing. The combined effect of PRRSV-elicited MIA and Poly(I:C) immune challenge further sensitized the pigs to behavior disruption across sexes including changes in sternal and lateral laying, walking, lethargy, and touching incidence. Notably, the effects of Poly(I:C) immune challenge alone on behaviors tended to be more extreme in males, whereas the effects of Poly(I:C) following MIA tended to be more extreme in females. Our findings demonstrate that MIA and Poly(I:C) affected behaviors, and the viral mimetic effects shortly after injection can offer insights into the prolonged effect of postnatal viral infections on feeding, social interactions and health status. Management practices that reduce the likelihood of gestational diseases and accommodate for behavioral disruptions in the offspring can minimize the impact of MIA.

Toll-like receptor 3 (TLR3)-mediated apoptotic changes in cancer cells are well-documented, and hence, several synthetic ligands of TLR3 are being used for adjuvant therapy, but there are reports showing a contradictory effect of TLR3 signaling, which include our previous report that had shown cell proliferation following surface localization of TLR 3. However, the underlying mechanism of cell surface localization of TLR3 and subsequent cell proliferation lacks clarity. This study addresses the TLR3 ligand-mediated signaling cascade that regulates a proliferative effect in breast cancer cells (MDA-MB-231 and T47D) challenged with TLR3 ligand in the presence of myeloid differentiation primary response 88 (MyD88) inhibitor. Evidences were obtained using immunoblotting, coimmunoprecipitation, confocal microscopy, immunocytochemistry, ELISA, and flow cytometry. Results had revealed that TLR3 ligand treatment significantly enhanced breast cancer cell proliferation marked by an upregulated expression of cyclinD1, but the same was suppressed by the addition of MyD88 inhibitor. Also, expression of interleukin 1 receptor-associated kinase 1 (IRAK1)-TNF receptor-associated factor 6 (TRAF6)-transforming growth factor beta-activated kinase 1 (TAK1) was altered in the given TLR3-signaling pathway. Inhibition of MyD88 disrupted the downstream adaptor complex and mediated signaling through the TLR3-MyD88-NF-κB (p65)-IL-6-cyclin D1 pathway. TLR3-mediated alternative signaling of the TLR3-MyD88-IRAK1-TRAF6-TAK1-TAB1-NF-κB axis leads to upregulation of IL6 and cyclin D1. This response is hypothesized to be via the MyD88 gateway that culminates in the proliferation of breast cancer cells. Overall, this study provides first comprehensive evidence on the involvement of canonical signaling of TLR3 using MyD88-cyclin D1-mediated breast cancer cell proliferation. The findings elucidated herein will provide valuable insights into understanding the TLR3-mediated adjuvant therapy in cancer.

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A+ and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ+&TNFα+, IL-17A+ and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ+ and IFNγ+&TNFα+, and H1N1-specific IL-17A+ T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ+ CTLs and H1N1-specific IFNγ+ T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ+&TNFα+ CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ+&TNFα+, and IL-17A+ phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks (H3K4me3, H3K4me1, H3K27ac and H3K27me3), we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C).

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to −20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Background: Altered white matter connectivity, as evidenced by pervasive microstructural changes in myelination and axonal integrity in neuroimaging studies, has been implicated in the development of autism spectrum disorder (ASD) and related neurodevelopmental conditions such as schizophrenia. Despite an increasing appreciation that such white matter disconnectivity is linked to social behavior deficits, virtually no etiologically meaningful myelin-related genes have been identified in oligodendrocytes, the key myelinating cells in the CNS, to furnish an account on the causes. The impact of neurodevelopmental perturbations during pregnancy such as maternal immune activation (MIA) on these genes in memory-related neural networks has not been experimentally scrutinized. Methods: In this study, a mouse model of MIA by the viral dsRNA analog poly(I:C) was employed to mimic the effects of inflammation during pregnancy. Transcriptional expression levels of selected myelin- or oligodendroglia-related genes implicated in schizophrenia or ASD development were analyzed by in situ hybridization (ISH) and quantitative real-time PCR (qRT-PCR) with brain samples from MIA and control groups. The analysis focused on SOX-10 (SRY-related HMG-box 10), MAG (myelin-associated glycoprotein), and Tf (transferrin) expression in the hippocampus and the surrounding memory-related cortical regions in either hemisphere. Results: Specifically, ISH reveals that in the brain of prenatal poly(I:C)-exposed mouse offspring in the MIA model (gestation day 9), mRNA expression of the genes SOX10, MAG and Tf were generally reduced in the limbic system including the hippocampus, retrosplenial cortex and parahippocampal gyrus on either side of the hemispheres. qRT-PCR further confirms the reduction of SOX10, MAG, and Tf expression in the medial prefrontal cortex, sensory cortex, amygdala, and hippocampus. Conclusions: Our present results provide direct evidence that prenatal exposure to poly(I:C) elicits profound and long-term changes in transcript level and spatial distribution of myelin-related genes in multiple neocortical and limbic regions, notably the hippocampus and its surrounding memory-related neural networks. Our work demonstrates the potential utility of oligodendroglia-related genes as biomarkers for modeling neurodevelopmental disorders, in agreement with the hypothesis that MIA during pregnancy could lead to compromised white matter connectivity in ASD.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-&alpha, and IL-1&szlig, cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFN&gamma, secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Peidi Yin,1 Huifeng Li,1 Chao Ke,2 Guangxu Cao,1 Xiaoqian Xin,3 Junjiao Hu,4 Xiangran Cai,4 Lingfeng Li,1 Xiaowen Liu,3 Bin Du1 1Department of Pathology, School of Medicine, Jinan University, Guangzhou 510632, People’s Republic of China; 2Department of Neurosurgery/Neuro-Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, People’s Republic of China; 3Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, People’s Republic of China; 4Medical Imaging Center, The First Affiliated Hospital, Jinan University, Guangzhou 510630, People’s Republic of ChinaCorrespondence: Xiaowen Liu; Bin Du Email xwliu231@jnu.edu.cn; tdubin@jnu.edu.cnPurpose: Some chemotherapeutics have been shown to induce both the release of damage-associated molecular patterns (DAMPs) and the production of type I interferon (IFN-I), leading to immunogenic cell death (ICD). However, the standard chemotherapy drug for glioma, temozolomide (TMZ), cannot induce ICD as it cannot activate IFN-I signaling. Moreover, inefficient delivery of immunostimulants across the blood–brain barrier (BBB) is the main obstacle to overcome in order to induce local immune responses in the brain.Methods: A new oligonucleotide nanoformulation (Au@PP)/poly(I:C)) was constructed by coating gold nanoparticles (AuNPs) with methoxypolyethylene glycol (mPEG)-detachable (d)-polyethyleneimine (PEI) (Au@PP) followed by inducing the formation of electrostatic interactions with polyinosinic-polycytidylic acid (poly(I:C)). Intracranial GL261 tumor-bearing C57BL/6 mice were used to explore the therapeutic outcomes of Au@PP/poly(I:C) plus TMZ in vivo. The anti-tumor immune response in the brain induced by this treatment was analyzed by RNA sequencing and immunohistochemical analyses.Results: Au@PP/poly(I:C) induced IFN-I production after endocytosis into glioma cells in vitro. Additionally, Au@PP/poly(I:C) was efficiently accumulated in the glioma tissue after intranasal administration, which allowed the nanoformulation to enter the brain while bypassing the BBB. Furthermore, Au@PP/poly(I:C) plus TMZ significantly improved the overall survival of the tumor-bearing mice compared with group TMZ only. RNA sequencing and immunohistochemical analyses revealed efficient immune response activation and T lymphocyte infiltration in the Au@PP/poly(I:C) plus TMZ group.Conclusion: This study demonstrates that intranasal administration of Au@PP/poly(I:C) combined with TMZ induces ICD, thereby stimulating an in situ immune response to inhibit glioma growth.Keywords: AuNPs, poly(I:C), intranasal administration, immunogenic cell death, in situ immune response activation

The prognostic value of Toll-like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early stage non-small-cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3&rsquo, s favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase-3 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re-activate local innate immune response.

Polyinosinic:polycytidylic acid (poly[I:C]) can stimulate Toll-like receptor 3 (TLR3) signaling pathways. In this study, DF-1 cells were treated with poly(I:C) at various concentrations and time points to examine the comparative expression patterns of innate immune response genes. The viability of DF-1 cells decreased from 77.41% to 38.68% when cells were treated different dose of poly(I:C) from 0.1 µg/mL to 100 µg/mL for 24 h respectively. The expressions of TLR3, TLR4, TLR7, TLR15, TLR21, IL1B, and IL10 were increased in dose- and time-dependent manners by poly(I:C) treatment. On the contrary, the expression patterns of interferon regulatory factors 7 (IRF7), Jun proto-oncogene, AP-1 transcription factor subunit (JUN), Nuclear Factor Kappa B Subunit 1 (NF-κB1), and IL8L2 were varied; IRF7 and IL8L2 were increasingly expressed whereas the expressions of JUN and NF-κB1 were decreased in a dose-dependent manner after they were early induced. In time-dependent analysis, IRF7 expression was significantly upregulated from 3 h to 24 h, whereas JUN and NF-κB1 expressions settled down from 6 h to 24 h after poly(I:C) treatment although they were induced at early time from 1 h to 3 h. Poly(I:C) treatment rapidly increased the expression of IL8L2 from 3 h to 6 h with a plateau at 6 h and then the expression of IL8L2 was dramatically decreased until 24 h after poly(I:C) treatment although the expression level was still higher than the non-treated control. These results may provide the basis for understanding host response to viral infection and its mimicry system in chickens.

Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3 L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2 weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4 weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1β and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli., Highlights • A robust protocol for the generation of high numbers of neonatal dendritic cells. • IL-3 and IL-6 are crucial for the proliferation of cord blood CD34+ progenitors. • Neonatal DCs have a higher inflammatory potential when exposed to viral stimuli. • LPS induces higher release of IL-10 in neonatal DCs compared to Poly(I:C).

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools to study neuronal and behavioral dysfunctions in relation to infection-mediated neurodevelopmental disorders. One of the most widely used MIA models is based on gestational administration of poly(I:C) (= polyriboinosinic-polyribocytdilic acid), a synthetic analog of double-stranded RNA that induces a cytokine-associated viral-like acute phase response. The effects of poly(I:C)-induced MIA on phenotypic changes in the offspring are known to be influenced by various factors, including the precise prenatal timing, genetic background, and immune stimulus intensity. Thus far, however, it has been largely ignored whether differences in the basic type of laboratory housing can similarly affect the outcomes of MIA models. Here, we examined this possibility by comparing the poly(I:C)-based MIA model in two housing systems that are commonly used in preclinical mouse research, namely the open cage (OC) and individually ventilated cage (IVC) systems. Pregnant C57BL6/N mice were kept in OCs or IVCs and treated with a low (1 mg/kg, i.v.) or high (5 mg/kg, i.v.) dose of poly(I:C), or with control vehicle solution. MIA or control treatment was induced on gestation day (GD) 9 or 12, and the resulting offspring were raised and maintained in OCs or IVCs until adulthood for behavioral testing. An additional cohort of dams was used to assess the influence of the different caging systems on poly(I:C)-induced cytokine and stress responses in the maternal plasma. Maternal poly(I:C) administration on GD9 caused a dose-dependent increase in spontaneous abortion in IVCs but not in OCs, whereas MIA in IVC systems during a later gestational time-point (GD12) did not affect pregnancy outcomes. Moreover, the precise type of caging system markedly affected maternal cytokines and chemokines at basal states and in response to poly(I:C) and further influenced the maternal levels of the stress hormone, corticosterone. The efficacy of MIA to induce deficits in working memory, social interaction, and sensorimotor gating in the adult offspring was influenced by the different housing conditions, the dosing of poly(I:C), and the precise prenatal timing. Taken together, the present study identifies the basic type of caging system as a novel factor that can confound the outcomes of MIA in mice. Our findings thus urge the need to consider and report the kind of laboratory housing systems used to implement MIA models. Providing this information seems pivotal to yield reproducible results in these models.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.

Summary The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-β and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing., Graphical Abstract, Highlights • Poly(I:C) activation of tissue-resident MSC leads to a time-dependent polarization • MSC early inflammatory response, via IFN-α/β, activates NK cells • MSC late regulatory response induces senescent-like NK cells via TGF-β and IL-6 • Senescent-like NK cell feedback to MSCs promotes survival and pro-angiogenic activity, Brandau et al. stimulated MSCs of the nasal mucosa with poly(I:C), a compound which mimics viral RNA. Although resting MSCs are thought to inhibit NK cell function, stimulated MSCs exerted a time-dependent biphasic response, which either promoted NK cell effector function (early response) or induced a senescent-like process (late response). The data suggest a model of time-dependent MSC polarization and innate immune interaction.

TLR3 belong to the Toll-like receptors family, it is mainly expressed on immune cells where it senses pathogen-associated molecular patterns and initiates innate immune response. TLR3 agonist poly(I:C) was developed to mimic pathogens infection and boost immune system activation to promote anti-cancer therapy. Accordingly, TLR agonists were included in the National Cancer Institute list of immunotherapeutic agents with the highest potential to cure cancer. Besides well known effects on immune cells, poly(I:C) was also shown, in experimental models, to directly induce apoptosis in cancer cells expressing TLR3. This review presents the current knowledge on the mechanism of poly(I:C)-induced apoptosis in cancer cells. Experimental evidences on positive or negative regulators of TLR3-mediated apoptosis induced by poly(I:C) are reported and strategies are proposed to successfully promote this event in cancer cells. Cancer cells apoptosis is an additional arm offered by poly(I:C), besides activation of immune system, for the treatment of various type of cancer. A further dissection of TLR3 signaling would contribute to greater resolution of the critical steps that impede full exploitation of the poly(I:C)-induced apoptosis. Experimental evidences about negative regulator of poly(I:C)-induced apoptotic program should be considered in combinations with TLR3 agonists in clinical trials.

In the central nervous system, viral infection can induce inflammation by up-regulating pro-inflammatory mediators that contribute to enhanced infiltration of immune cells into the central nervous areas. Celastrol is known to exert various regulatory functions, including anti-microbial activities. In this study, we investigated the regulatory effects and the mechanisms of action of celastrol against astrocytes activated with polyinosinic-polycytidylic acid (poly(I:C)), a synthetic dsRNA, as a model of pro-inflammatory mediated responses. Celastrol significantly inhibited poly(I:C)-induced expression of adhesion molecules, such as ICAM-1/VCAM-1, and chemokines, such as CCL2, CXCL8, and CXCL10, in CRT-MG human astroglioma cells. In addition, celastrol significantly suppressed poly(I:C)-induced activation of JNK MAPK and STAT1 signaling pathways. Furthermore, celastrol significantly suppressed poly(I:C)-induced activation of the NF-κB signaling pathway. These results suggest that celastrol may exert its regulatory activity by inhibiting poly(I:C)-induced expression of pro-inflammatory mediators by suppressing activation of JNK MAPK-STAT1/NF-κB in astrocytes. [BMB Reports 2017; 50(1): 25-30].

Maternal immune activation (MIA) models that are based on administration of the viral mimetic, poly(I:C), are widely used as experimental tools to study neuronal and behavioral dysfunctions in relation to immune-mediated neurodevelopmental disorders and mental illnesses. Evidence from investigations in non-pregnant rodents suggests that different poly(I:C) products can vary in terms of their immunogenicity, even if they are obtained from the same vendor. The present study aimed at extending these findings to pregnant mice, while also controlling various poly(I:C) products for potential contamination with lipopolysaccharide (LPS). We found significant variability between different batches of poly(I:C) potassium salt obtained from the same vendor (Sigma-Aldrich) in terms of the relative amount of dsRNA fragments in the high molecular weight range (1000-6000 nucleotides long) and with regards to their effects on maternal thermoregulation and immune responses in maternal plasma, placenta and fetal brain. Batches of poly(I:C) potassium salt containing larger amounts of high molecular weight fragments induced more extensive effects on thermoregulation and immune responses compared to batches with minimal amounts of high molecular weight fragments. Consistent with these findings, poly(I:C) enriched for high molecular weight dsRNA (HMW) caused larger maternal and placental immune responses compared to low molecular weight (LMW) poly(I:C). These variable effects were unrelated to possible LPS contamination. Finally, we found marked variability between different batches of the poly(I:C) potassium salt in terms of their effects on spontaneous abortion rates. This batch-to-batch variability was confirmed by three independent research groups using distinct poly(I:C) administration protocols in mice. Taken together, the present data confirm that different poly(I:C) products can induce varying immune responses and can differentially affect maternal physiology and pregnancy outcomes. It is therefore pivotal that researchers working with poly(I:C)-based MIA models ascertain and consider the precise molecular composition and immunogenicity of the product in use. We recommend the establishment of reference databases that combine phenotype data with empirically acquired quality information, which can aid the design, implementation and interpretation of poly(I:C)-based MIA models.

Maternal immune activation (MIA), be it triggered by infectious or noninfectious stimuli, is implicated in various psychiatric and neurological disorders with developmental etiologies. Its consequences on the offspring's mental health are heterogeneous and influenced by a number of factors shaping the specificity and/or severity of pathological outcomes. There is also a substantial degree of resilience to MIA, which determines the extent to which offspring are protected from developing neurodevelopmental sequelae. This review provides a synopsis of the plausible sources that account for the heterogeneous outcomes of MIA and discusses key factors that are critical for establishing neurodevelopmental resilience and susceptibility to this early-life adversity.

Intermitted fasting and other forms of calorie restriction are increasingly demonstrated to exert potential health benefits. Interestingly, restricted feeding is also able to mitigate sickness in response to bacterial factors stimulating Toll-like receptor 4 (TLR4). However, little is known about how fasting modifies the activity of virus-associated molecular patterns. We therefore analyzed the impact of an intermittent fasting (IF) regimen on the immune and behavioral response to the TLR3 agonist and viral mimic polyinosinic:polycytidylic acid [Poly(I:C)] in mice. The effects of intraperitoneally injected Poly(I:C) (12 mg/kg) on plasma and cerebral cytokine expression and behavior (locomotion, exploration, and ingestion) were examined in male C57BL/6N mice under control conditions and following a 9 days period of intermittent (alternate day) fasting (IF). Poly(I:C) increased the circulating levels of cytokines (TNF-α, MCP-1, IL-6, IL-10, IFN-α, IFN-γ), an effect amplified by IF. In addition, IF aggravated sickness behavior in response to Poly(I:C), while cerebral cytokine expression was enhanced by application of Poly(I:C) in the absence of a significant effect of IF. Furthermore, IF augmented the expression of neuropeptide Y (NPY) mRNA in the hypothalamus and increased the plasma levels of corticosterone, while Poly(I:C) had little effect on these readouts. Our data show that IF does not abate, but exaggerates the immune and sickness response to the viral mimic Poly(I:C). This adverse effect of IF occurs despite increased hypothalamic NPY expression and enhanced plasma corticosterone. We therefore propose that the effects of IF on the immune and behavioral responses to viral and bacterial factors are subject to different neuronal and neuroendocrine control mechanisms.

Macrophage plasticity is the ability of mononuclear phagocytes to change phenotype, function, and genetic reprogramming upon encounter of specific local stimuli. In the tumor microenvironment, Tumor-Associated Macrophages (TAMs) acquire an immune- suppressive and tumor-promoting phenotype. With the aim to re-educate TAMs to anti- tumor effectors, in this study, we used two immunestimulatory compounds: the TLR7 agonist Imiquimod (IMQ) and the TLR3 agonist Poly(I:C). To better mimic in vitro the response of TAMs, we used Tumor-Conditioned Macrophages (TC-Mφ) differentiated in the presence of tumor cell supernatants. Our results show that TC-Mφrespond differently from conventional M2-polarized macrophages. Upon stimulation with IMQ, TC-Mφdid not upregulate major histocompatibility complex (MHC II) molecules and unexpectedly expressed increased CD206. With both compounds, TC-Mφproduced higher levels of inflammatory cytokines than M2 macrophages. IMQ and Poly(I:C) differed in the types of regulated genes and secreted mediators. Reflecting their signaling pathways, only IMQ significantly induced IL-1βand IL-6, while only Poly(I:C) stimulated CXCL10, and both upregulated CCL5. Of note, using a novel cytotoxicity assay, Poly(I:C), but not IMQ, was effective in triggering the cytotoxic activity of TC-Mφagainst cancer cells. Overall, the results demonstrate that Poly(I:C) stimulation of TC-Mφis superior than IMQ in terms of macrophage re-education toward antitumor effectors.

Background Important insight into the mechanisms through which gene-environmental interactions cause schizophrenia can be achieved through preclinical studies combining prenatal immune stimuli with disease-related genetic risk modifications. Accumulating evidence associates JNK signalling molecules, including MKK7/MAP2K7, with genetic risk. We tested the hypothesis that Map2k7 gene haploinsufficiency in mice would alter the prenatal immune response to the viral mimetic polyriboinosinic-polyribocytidylic acid (polyI:C), specifically investigating the impact of maternal versus foetal genetic variants. Methods PolyI:C was administered to dams (E12.5), and cytokine/chemokine levels were measured 6 h later, in maternal plasma, placenta and embryonic brain. Results PolyI:C dramatically elevated maternal plasma levels of most cytokines/chemokines. Induction of IL-1β, IL-2, IL-10, IL-12, TNF-α and CXCL3 was enhanced, while CCL5 was suppressed, in Map2k7 hemizygous (Hz) dams relative to controls. Maternal polyI:C administration also increased embryonic brain chemokines, influenced by both maternal and embryonic genotype: CCL5 and CXCL10 levels were higher in embryonic brains from Map2k7 dams versus control dams; for CCL5, this was more pronounced in Map2k7 Hz embryos. Placental CXCL10 and CXCL12 levels were also elevated by polyI:C, the former enhanced and the latter suppressed, in placentae from maternal Map2k7 Hzs relative to control dams receiving polyI:C. Conclusions The results demonstrate JNK signalling as a mediator of MIA effects on the foetus. Since both elevated CXCL10 and supressed CXCL12 compromise developing GABAergic interneurons, the results support maternal immune challenge contributing to schizophrenia-associated neurodevelopmental abnormalities. The influence of Map2k7 on cytokine/chemokine induction converges the genetic and environmental aspects of schizophrenia, and the overt influence of maternal genotype offers an intriguing new insight into modulation of embryonic neurodevelopment by genetic risk. Electronic supplementary material The online version of this article (10.1186/s12974-019-1408-5) contains supplementary material, which is available to authorized users.

Host defense peptides (HDPs) show both antimicrobial and immunomodulatory properties making them important mediators of the host immune system. In humans but also in pigs many HDPs have been identified and important families such as cathelicidins and defensins have been established. In our study, we assessed: (i) the potential interactions that could occur between three peptides (LL37, PR39, and synthetic innate defense regulator (IDR)-1002) and a common TLR ligand called poly(I:C); (ii) the impact of selected peptides on the response of alveolar macrophage (AM) to poly(I:C) stimulation; (iii) the anti-porcine respiratory and reproductive syndrome virus (PRRSV) properties of the peptides; and (iv) their adjuvant potential in a PRRSV challenge experiment after immunization with different vaccine formulations. The results are as following: LL37, PR39, and IDR-1002 were able to interact with poly(I:C) using an agarose gel migration assay. Then, an alteration of AM's response to poly(I:C) stimulation was observed when the cells were co-stimulated with LL37 and IDR-1002. Regarding the anti-PRRSV potential of the peptides only LL37 showed a PRRSV inhibition in infected AM as well as precision cut lung slices (PCLS). However, in our conditions and despite their immunomodulatory properties, neither LL37 nor IDR-1002 showed any convincing potential as an adjuvant when associated to killed PRRSV in a challenge experiment. In conclusion, both antiviral and immunomodulatory properties could be identified for LL37, only immunomodulatory properties for IDR-1002, and both peptides failed to improve the immune response consecutive to an immunization with a killed vaccine in a PPRSV challenge experiment. However, further studies are needed to fully decipher and explain differences between peptide properties.

ARTICLE IN PRESS; International audience; At the interface of the environment and the nervous system, the olfactory mucosa (OM) is a privileged pathway for environmental toxicants and pathogens towards the central nervous system. The OM is known to produce antimicrobial and immunological components but the mechanisms of action of the immune system on the OM remain poorly explored. IL-17c is a potent mediator of respiratory epithelial innate immune responses, whose receptors are highly expressed in the OM of mice. We first characterized the presence of the IL-17c and its receptors in the OM. While IL-17c was weakly expressed in the control condition, it was strongly expressed in vivo after intranasal administration of polyinosinic–polycytidylic (Poly I:C), a Toll Like Receptor 3 agonist, mimicking a viral infection. Using calcium imaging and electrophysiological recordings, we found that IL-17c can effectively activate OM cells through the release of ATP. In the longer term, intranasal chronic instillations of IL-17c increased the cellular dynamics of the epithelium and promoted immune cells infiltrations. Finally, IL-17c decreased cell death induced by Poly(I:C) in an OM primary culture. The OM is thus a tissue highly responsive to immune mediators, proving its central role as a barrier against airway pathogens.