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The economic importance of lumpfish ( Cyclopterus lumpus ) is increasing, but several aspects of its immune responses are not well understood. To discover genes and mechanisms involved in the lumpfish antiviral response, fish were intraperitoneally injected with either the viral mimic polyinosinic:polycytidylic acid [poly(I:C)] or phosphate-buffered saline (PBS; vehicle control), and head kidneys were sampled 24 hours post-injection (hpi) for transcriptomic analyses. RNA sequencing (RNA-Seq) (adjusted p-value <0.05) identified 4,499 upregulated and 3,952 downregulated transcripts in the poly(I:C)-injected fish compared to the PBS-injected fish. Eighteen genes identified as differentially expressed by RNA-Seq were included in a qPCR study that confirmed the upregulation of genes encoding proteins with antiviral immune response functions (e.g., rsad2 ) and the downregulation of genes (e.g., jarid2b ) with potential cellular process functions. In addition, transcript expression levels of 12 members of the interferon regulatory factor (IRF) family [seven of which were identified as poly(I:C)-responsive in this RNA-Seq study] were analyzed using qPCR. Levels of irf1a , irf1b , irf2 , irf3 , irf4b , irf7 , irf8 , irf9 , and irf10 were significantly higher and levels of irf4a and irf5 were significantly lower in the poly(I:C)-injected fish compared to the PBS-injected fish. This research and associated new genomic resources enhance our understanding of the genes and molecular mechanisms underlying the lumpfish response to viral mimic stimulation and help identify possible therapeutic targets and biomarkers for viral infections in this species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Emam, Kumar, Eslamloo, Caballero-Solares, Hall, Xue, Paradis, Gendron, Santander and Rise.)

Neonate responses to pathogen-associated molecular patterns (PAMPS) differ from adults; such understanding is poor in Indian neonates, despite recognized significant infectious risk. Immune profiling analysis was undertaken of 10 secreted mediators contextualized with cellular source induced by six PAMPs in umbilical cord (CB; n = 21) and adult-blood (PBMC; n = 14) from a tertiary care hospital in South India. Differential cytokine expression analysis (minimum log2-fold difference; adj P-value < 0.05) identified bacterial PAMPs induced higher concentrations of IL-1β, IL-10, TNF-α in adults versus IL-8, GM-CSF, IFN-γ, and IL-2 in CB. CB responded to poly I:C and SARS-CoV-2 lysate with a dominant IL-8 response, whereas in PBMC, CXCL-10 dominated poly I:C, but not SARS-CoV-2, responses, highlighting potential IL-8 importance, in the absence of Type I Interferons, in antiviral CB immunity. Candida albicans was the only PAMP to uniformly induce higher secretion of effectors in CB. The predominant source of IL-8/IL-6/TNF-α/IL-1β in both CB and PBMC was polyfunctional monocytes and IFN-γ/IL-2/IL-17 from innate lymphocytes. Correlation matrix analyses revealed IL-8 to be the most differentially regulated, correlating positively in CB versus negatively in PBMC with IL-6, GM-CSF, IFN-γ, IL-2, consistent with more negatively regulated cytokine modules in adults, potentially linked to higher anti-inflammatory IL-10. Cord and adult blood from India respond robustly to PAMPs with unique effector combinations. These data provide a strong foundation to monitor, explore, mechanisms that regulate such immunity during the life course, an area of significant global health importance given infection-related infant mortality incidence., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

3-phosphoinositide-dependent protein kinase-1 (PDK-1) is a key kinase regulating the activity of the PI3K/AKT pathway and a major regulator of the AGC protein kinase family. It is essential in the physiological activities of cells, embryonic development, individual development and immune response. In this study, we have identified for the first time an analogue of PDK-1 in the most primitive vertebrate, lamprey, and named it PDK-1-like. The protein sequence similarity of lamprey PDK-1-like to human, mouse, chicken, African xenopus and zebrafish PDK-1 were 64.4 %, 64.5 %, 65.0 %, 61.3 % and 63.2 %, respectively. The phylogenetic tree showed that PDK-1-like of lamprey were located at the base of the vertebrate branch, in line with the trend of biological evolution. Meanwhile, homology analysis showed that PDK-1 proteins across species shared a conserved kinase structural domain and a Pleckstrin Homology (PH) domain. Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey PDK-1-like presented dramatic differences compared with jawed vertebrates. More importantly, qPCR analysis showed that PDK-1-like was widely expressed in lamprey. Its mRNA expression levels varied in response to different pathogenic stimuli, and its expression was generally up-regulated under Polyinosinic-Polycytidylic acid (Poly(I:C)) stimulation. Pearson's correlation analysis showed that PDK-1-like was involved in co-expressed with MyD88-independent TLR-3 pathway during the immune response of lamprey, instead of MyD88-dependent TLR-3 pathway. In summary, our composite results offer valuable clues to the origin and evolution of PDK-1, and imply that PDK-1 s are among the most ancestral immune regulators in vertebrates., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms., Competing Interests: Declaration of competing interest The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-β (IFN-β), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6'-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value., Competing Interests: Declaration of competing interest EŠ, GE, JTJ, GJ and GKP are employed by Statens Serum Institut, which is a non-profit government research facility, holding patents on the cationic adjuvant formulations (CAF®)., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (C p -C 52 ) and resolving cysteine (C R -C 173 ) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1 (+) /AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm&#95;classA&#95;rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Low molecular weight proteins, known as chemokines, facilitate the migration and localization of immune cells to the site of infection and injury. One of the first chemokines identified, CXCL8 functions as a key neutrophil activator, recruiting neutrophils to sites of inflammation. Several viral infections, including zoonotic coronaviruses and poxviruses, have been reported to induce the expression of CXCL8. Dromedary camels are known to harbor several potentially zoonotic pathogens, but critical immune molecules such as chemokines remain unidentified. We report here the identification of CXCL8 from the dromedary camel - the first chemokine identified from camelids. The complete dromedary CXCL8 cDNA sequence as well as the corresponding gene sequence from dromedary and two New World camelids - alpaca and llama were cloned. CXCL8 mRNA expression was relatively higher in PBMC, spleen, lung, intestine, and liver. Poly(I:C) and lipopolysaccharide stimulated CXCL8 expression in vitro, while interferon treatment inhibited it. In vitro infection with potentially zoonotic camelpox virus induced the expression of CXCL8 in camel kidney cells. Toxicological studies on camelids have been limited, and no biomarkers have been identified. Hence, we also evaluated CXCL8 mRNA expression as a potential biomarker to assess heavy metal toxicity in camel kidney cells in vitro. CXCL8 expression was increased after in vitro exposure to heavy metal compounds of cobalt and cadmium, suggesting potential utility as a biomarker for renal toxicity in camels. The results of our study demonstrate that camel CXCL8 plays a significant role in immunomodulatory and induced toxicity responses in dromedary camels., Competing Interests: Declaration of competing interests The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Members of the myxovirus resistance (Mx) protein family play an essential role in antiviral immunity. They are Dynamin-like GTPases, induced by interferons. In the current study, we have characterized two predicted MX genes (MX1 and MX2) from lumpfish (Cyclopterus lumpus L.), having 12 and 13 exons, respectively. Mx2 has two isoforms (Mx2-X1 and Mx2-X2) which differ in exon 1. The lumpfish Mx proteins contain an N-terminal Dynamin-like GTPase domain, the middle domain (MD) and GTPase effector domain (GED) characteristic for Mx proteins. Phylogenetic analyses grouped all the lumpfish Mx sequences in group 1, and synteny analyses showed that both genes were localized at chromosome 5 in proximity to the genes Tohc7, Atxn7 and Psmd6. In vitro stimulation experiment showed that both MX1 and MX2-X2 were highly upregulated upon exposure to poly(I:C), but not bacteria, 24 h post exposure, indicating their role in antiviral immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a)., Methods: The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations., Results: The full-length CDS of Labeo rohita STAT1a ( LrSTAT1a ) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a . Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106., Discussion: This study provides molecular insights into the STAT1a protein in Rohu ( Labeo rohita ) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Das, Panda, Pradhan, Raut, Kumari and Meena.)

Exosomes, extracellular vesicles (EVs) produced within cells, mediate both the disposal of intracellular waste and communication with distant cells, and they are involved in a variety of disease processes. Although disease modifications of exosome cargos have been well studied, it has been poorly investigated how disease processes, such as endoplasmic reticulum (ER) stress, affect EV production. We previously reported that adiponectin, an adipocyte-secreted salutary factor, increases systemic exosome levels through T-cadherin-mediated enhancement of exosome biogenesis. In the present study, we demonstrated that adiponectin/T-cadherin-dependent EV production was susceptible to ER stress and that low-dose tunicamycin significantly reduced EV production in the presence, but not in the absence, of adiponectin. Moreover, pharmacological or genetic activation of inositol-requiring enzyme 1α, a central regulator of ER stress, downregulated T-cadherin at the mRNA and protein levels as well as attenuated EV production. In addition, adiponectin/T-cadherin-independent EV production was attenuated under ER stress conditions. Repeated administration of tunicamycin to mice decreased circulating small EVs without decreasing tissue T-cadherin expression. Mechanistically, inositol-requiring enzyme 1α activation by silencing of the X-box binding protein 1 transcription factor upregulated the canonical interferon pathway and decreased EV production. The interferon pathway, when it was activated by polyinosinic-polycytidylic acid, also significantly attenuated EV production. Thus, we concluded that ER stress decreases exosome production through adiponectin/T-cadherin-dependent and -independent pathways., Competing Interests: Conflict of interest K. F. is an employee of Kowa Company Ltd. S. K. belongs to the endowed department by Takeda Pharmaceutical Company, Rohto Pharmaceutical Co, Ltd, Sanwa Kagaku Kenkyusho Co, Ltd, FUJI OIL HOLDINGS INC, and Kobayashi Pharmaceutical Co, Ltd. N. M. belongs to the endowed department by Kowa Company Ltd. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor infiltration by T cells profoundly affects cancer progression and responses to immunotherapy. However, the tumor immunosuppressive microenvironment can impair the induction, trafficking, and local activity of antitumor T cells. Here, we investigated whether intratumoral injection of virus-derived peptide epitopes could activate preexisting antiviral T cell responses locally and promote antitumor responses or antigen spreading. We focused on a mouse model of cytomegalovirus (CMV), a highly prevalent human infection that induces vigorous and durable T cell responses. Mice persistently infected with murine CMV (MCMV) were challenged with lung (TC-1), colon (MC-38), or melanoma (B16-F10) tumor cells. Intratumoral injection of MCMV-derived T cell epitopes triggered in situ and systemic expansion of their cognate, MCMV-specific CD4 + or CD8 + T cells. The MCMV CD8 + T cell epitopes injected alone provoked arrest of tumor growth and some durable remissions. Intratumoral injection of MCMV CD4 + T cell epitopes with polyinosinic acid:polycytidylic acid (pI:C) preferentially elicited tumor antigen-specific CD8 + T cells, promoted tumor clearance, and conferred long-term protection against tumor rechallenge. Notably, secondary proliferation of MCMV-specific CD8 + T cells correlated with better tumor control. Importantly, intratumoral injection of MCMV-derived CD8 + T cell-peptide epitopes alone or CD4 + T cell-peptide epitopes with pI:C induced potent adaptive and innate immune activation of the tumor microenvironment. Thus, CMV-derived peptide epitopes, delivered intratumorally, act as cytotoxic and immunotherapeutic agents to promote immediate tumor control and long-term antitumor immunity that could be used as a stand-alone therapy. The tumor antigen-agnostic nature of this approach makes it applicable across a broad range of solid tumors regardless of their origin.

Gestational maternal immune activation (MIA) in mice induces persistent brain microglial activation and a range of neuropathologies in the adult offspring. Although long-term phenotypes are well documented, how MIA in utero leads to persistent brain inflammation is not well understood. Here, we found that offspring of mothers treated with polyriboinosinic–polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood–brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte–endothelium coupling in cerebral blood vessels and increased microglial activation were found in fetal and 1- and 6-mo-old offspring brains. The long-lasting disruptions result from abnormal prenatal BBB formation, driven by increased proliferation of cyclooxygenase-2 (COX2; Ptgs2)-expressing microglia in fetal brain parenchyma and perivascular spaces. Targeted deletion of the Ptgs2 gene in fetal myeloid cells or treatment with the inhibitor celecoxib 24 h after immune activation prevented microglial proliferation and disruption of BBB formation and function, showing that prenatal COX2 activation is a causal pathway of MIA effects. Thus, gestational MIA disrupts fetal BBB formation, inducing persistent BBB dysfunction, which promotes microglial overactivation and behavioral alterations across the offspring life span. Taken together, the data suggest that gestational MIA disruption of BBB formation could be an etiological contributor to neuropsychiatric disorders.

Subcellular localization analysis implicated that CiPRMT6 was mainly located in the nucleus, with a small part of them located in the cytoplasm. PRMT6, namely protein arginine methyltransferase 6, was first identified and demonstrated to catalyze the methylation of arginine residue on the chromatin histones in mammals. Mammalian PRMT6 usually acts as an arginine methyltransferase in the nucleus, but induces antiviral innate immune response in the cytoplasm. Nowadays, there have been few reports about PRMT6 in teleost. In this study, we investigated the potential molecular mechanisms underlying the interaction of PRMT6 expression and IFN1 response in grass carp. We first cloned and identified a grass carp PRMT6 (named CiPRMT6, MN781672.1), which is 1068bp in length encoding a deduced polypeptide of 355 amino acids. In CIK cell, CiPRMT6 expression was up-regulated upon stimulation with poly (I:C); while overexpression of PRMT6 suppressed the promoter activity of grass carp IFN1 and reduced the phosphorylation of IRF3; however, the amount of PRMT6 mutant (lack of methyltransferase domain) was increased in the cytoplasm. Our results also showed that grass carp PRMT6 and IRF3 (but not TBK1) were co-located and bound to each other in the cytoplasm. The binding of CiPRMT6 to IRF3 impairs the interaction between TBK1 and IRF3, indicating that CiPRMT6 is a negative regulator for IFN1 expression through TBK1-IRF3 signaling pathway in grass carp. In conclusion, we identified that CiPRMT6 negatively regulated IFN1 expression by inhibiting the TBK1-IRF3 interaction as well as IRF3 phosphorylation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Maternal immune activation (MIA) with poly(I:C) is a preclinical paradigm for schizophrenia and autism research. Methodological variations, including poly(I:C) molecular weight, contribute to inconsistencies in behavioural and molecular outcomes. We established in Wistar rats that 4 mg/kg high molecular weight (HMW)-poly(I:C) on GD19 induces maternal sickness, smaller litters and maternal elevations of serum cytokines, including increases in monocyte chemoattractants. In adult offspring, we found that males have higher serum cytokines than females, and MIA did not alter peripheral cytokines in either sex. Our study will contribute to the effective use of the MIA model to elucidate the neurobiology of neurodevelopmental disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Targeting antigens to dendritic cells represent a promising method for enhancing immune responses against specific antigens. However, many studies have focused on systemic delivery (intravenous or intraperitoneally) of targeted antigen, approaches that are not easily transferable to humans. Here we evaluate the efficacy of an influenza vaccine targeting Xcr1 + cDC1 administered by intranasal immunization. Intranasal delivery of antigen fused to the chemokine Xcl1, the ligand of Xcr1, resulted in specific uptake by lung CD103 + cDC1. Interestingly, intranasal immunization with influenza A/PR/8/34 haemagglutinin (HA) fused to Xcl1, formulated with poly(I:C), resulted in enhanced induction of antigen-specific IFNγ + CD4 + and IFNγ + CD8 + T cell responses in lung compared non-targeted anti-NIP-HA (αNIP-HA). Induction of antibody responses was, however, similar in Xcl1-HA and αNIP-HA immunized mice, but significantly higher than in mice immunized with monomeric HA. Both Xcl1-HA and αNIP-HA vaccines induced full protection when mice were challenged with a lethal dose of influenza PR8 virus, reflecting the strong induction of HA-specific antibodies. Our results demonstrate that i.n. delivery of Xcl1-HA is a promising vaccine strategy for enhancing T cell responses in addition to inducing strong antibody responses., (© 2021 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

Background: Innate immunity, armed with pattern recognition receptors including Toll-like receptors (TLR), is critical for immune cell activation and the connection to anti-microbial adaptive immunity. However, information regarding the impact of age on the innate immunity in response to SARS-CoV2 adenovirus vector vaccines and its association with specific immune responses remains scarce., Methods: Fifteen subjects between 25-35 years (the young group) and five subjects between 60-70 years (the older adult group) were enrolled before ChAdOx1 nCoV-19 (AZD1222) vaccination. We determined activation markers and cytokine production of monocyte, natural killer (NK) cells and B cells ex vivo stimulated with TLR agonist (poly (I:C) for TLR3; LPS for TLR4; imiquimod for TLR7; CpG for TLR9) before vaccination and 3-5 days after each jab with flow cytometry. Anti-SARS-CoV2 neutralization antibody titers (surrogate virus neutralization tests, sVNTs) were measured using serum collected 2 months after the first jab and one month after full vaccination., Results: The older adult vaccinees had weaker vaccine-induced sVNTs than young vaccinees after 1 st jab (47.2±19.3% vs. 21.2±22.2%, p value<0.05), but this difference became insignificant after the 2 nd jab. Imiquimod, LPS and CpG strongly induced CD86 expression in IgD + CD27 - naïve and IgD - CD27 + memory B cells in the young group. In contrast, only the IgD + CD27 - naïve B cells responded to these TLR agonists in the older adult group. Imiquimode strongly induced the CD86 expression in CD14 + monocytes in the young group but not in the older adult group. After vaccination, the young group had significantly higher IFN-γ expression in CD3 - CD56 dim NK cells after the 1 st jab, whilst the older adult group had significantly higher IFN-γ and granzyme B expression in CD56 bright NK cells after the 2 nd jab (all p value <0.05). The IFN-γ expression in CD56 dim and CD56 bright NK cells after the first vaccination and CD86 expression in CD14 + monocyte and IgD - CD27 - double-negative B cells after LPS and imiquimod stimulation correlated with vaccine-induced antibody responses., Conclusions: The innate immune responses after the first vaccination correlated with the neutralizing antibody production. Older people may have defective innate immune responses by TLR stimulation and weak or delayed innate immune activation profile after vaccination compared with young people., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shen, Yen, Fu, Cheng, Shen, Chang, Cheng, Liu, Chang, Chen, Ko and Shieh.)

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.

A promising strategy for controlling animal brucellosis is vaccination with commercial vaccine strains (Brucella melitensis Rev.1 and Brucella abortus RB51). Owing to safety concerns associated with these vaccines, developing a more effective and safe vaccine is essential. In this study, we examined the capacity of BhuA, 7α-HSDH or FliC antigens in the presence or absence of adjuvant in eliciting immune responses against brucellosis. After cloning, expression and purification, these proteins were used to examine immunologic responses. All immunized mice induced a vigorous IgG, with a predominant IgG2a response. Moreover, splenocytes of immunized mice proliferated and produced IL-2 and IFN-γ, suggesting the induction of cellular immunity. The high IgG2a/IgG1 ratio and IL-2 and IFN-γ indicated a Th1-oriented immune response in test groups. BhuA-, 7α-HSDH- or FliC- poly I:C formulations were the most effective at inducing Th1 immune response compared to groups immunized with naked proteins. Immunization with proteins protected mice against B. melitensis 16M and B. abortus 544. The proteins in adjuvant induced higher levels of protection than proteins only and exhibited similar degree of protection to live attenuated vaccines. Our results, for first time, introduced five potential candidates for subunit vaccine development against B. melitensis and B. abortus infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

IκB kinase β (IKKβ), a catalytic subunit of the IKK complex, is involved in a wide array of biological processes, particularly in inflammation and innate immunity. Although extensive studies have been carried out to explore the roles of mammalian IKKβs in innate immune response, the function of IKKβ in avian innate immunity is largely unknown. Here, we cloned and characterized the duck IKKβ (duIKKβ) gene for the first time. DuIKKβ encoded 755 amino acids and displayed high sequence similarity to pseudopodoces and haliaeetus IKKβs. DuIKKβ transcripts were widely distributed in all tested tissues, especially with high expression in the thymus and bursa of Fabricius. Overexpression of duIKKβ promoted NF-κB activation and initiated the downstream cytokines expression including IFN-β, ZAP, PKR, IL-8, and CCL5 in duck embryo fibroblasts. Furthermore, knockdown of endogenous duIKKβ significantly reduced LPS-, poly(I:C)- or SeV-induced NF-κB activation. Finally, we demonstrated that duIKKβ showed antiviral activity against Duck Tembusu virus infection. Our findings provide insights into the roles of duIKKβ in avian innate immunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) play an important role in the host defense system of shrimps. In this study, an Arasin-like peptide, named as LvArasin-like, was identified from the hemocytes of the pacific white shrimp, Litopenaeus vannamei. The complete open reading frame (ORF) of LvArasin-like was 213 bp, encoding 70 amino acid residues with a predicted molecular mass of 5.68 kDa and a theoretical isoelectric point (pI) of 6.73. The predicted peptide consisted of a signal peptide, an N-terminal Pro/Arg-rich domain, and a C-terminal cysteine-rich domain. LvArasin-like expression was most abundant in the gills and was up-regulated in hemocytes after LPS or Poly I:C injection as well as challenges by Vibrio parahaemolyticus or Staphylococcus aureus infection. In the heterologous expression system, LvArasin-like protein (rLvArasin-like) was recombinantly expressed in the forms of a dimer or both a monomer and dimer. The rLvArasin-like could directly bind to gram-positive and gram-negative bacteria and exhibited broad-spectrum antimicrobial activity towards them, with 50 % of minimal inhibitory concentrations (MIC50) of 6.25-50 μM. Moreover, dsRNA-mediated knockdown of LvArasin-like enhanced the susceptibility of shrimp to V. parahaemolyticus. In addition, the transcriptional level of LvArasin-like was downregulated when silencing of the transcription factors LvDorsal and LvRelish using RNAi in vivo. All of these results suggest that LvArasin-like is involved in host defense against bacterial infection. Therefore, it is a potential therapeutic agent for disease control in shrimp aquaculture., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Toll-like receptor 7 (TLR7) subfamily members are important pattern recognition receptors that participate in the recognition of pathogen-associated molecular patterns. In the present study, three TLR family members, OnTLR7, OnTLR8 and OnTLR9, were identified in the Nile tilapia Oreochromis niloticus. TLR7-, TLR8-and TLR9-deduced proteins have typical structural characteristics of TLRs, including Toll/interleukin-1 receptor (TIR), leucine-rich repeat (LRR) and transmembrane region (TM). OnTLR7, OnTLR8 and OnTLR9 were broadly expressed in all of the tissues tested, with the highest expression levels in the brain (TLR7) and spleen (TLR8 and TLR9). Moreover, the expression levels of OnTLR7, OnTLR8 and OnTLR9 were significantly increased in most tested tissues after Streptococcus agalactiae infection in vivo. After LPS stimulation, OnTLR7 and OnTLR9 mRNA expression levels were downregulated in the intestine and upregulated in the liver, spleen and kidney; however, OnTLR8 mRNA expression levels were upregulated in the kidney only after LPS stimulation for 5 d. After Poly I:C stimulation, OnTLR7 and OnTLR9 mRNA expression levels were upregulated in the intestine, liver, spleen and kidney, and the highest expression was found in the liver, while OnTLR8 mRNA expression levels were upregulated in the intestine, liver and kidney and downregulated in the spleen. Subcellular localization of OnTLR7, OnTLR8, and OnTLR9 in 293T cells showed that OnTLR9 was distributed in both the cytoplasm and nucleus while OnTLR8 and OnTLR7 were distributed mainly in the cytoplasm. Overexpression of OnTLR7, OnTLR8 and OnTLR9 in 293T cells had no significant effect on the activity of NF-κB, but they could significantly enhance MyD88-mediated NF-κB activity after cotransfection with MyD88. Pulldown assays showed that OnTLR7, OnTLR8, and OnTLR9 could interact with OnMyD88. Taken together, these results indicate that TLR7 subfamily genes play a role in the immune response to pathogen invasion of Nile tilapia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNβ or IFNλ increased PD-L1 and PD-L2, and IFNβ or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNβ, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells., Competing Interests: KM received an honorarium for educational lectures from AstraZeneca and GlaxoSmithKline Pharmaceuticals Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ogawa, Kan-o, Shiota, Fujita, Ishii, Fukuyama and Matsumoto.)

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8 + and CD4 + T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Research Question: In previous studies, we demonstrated that the human decidua and decidual stromal cells express high levels of CCL2 (chemokine (C-C motif) ligand 2, also known as monocyte chemotactic protein-1) and its receptor CCR2 (chemokine receptor 2). DSC-derived CCL2 interacts with CCR2 on DICs, causing the production and secretion of Th2-type cytokines, which promotes a Th2 bias at the maternal-foetal interface. Many pathogens may be present in the genital tract during pregnancy, but whether they affect immune regulation, especially Th2 regulation remains unknown. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that recognise specific components of microbes and certain host molecules and play an important role in the host innate immune response. We examined TLR expression and evaluated whether TLRs could affect CCL2 secretion and subsequently induce Th1/Th2 responses., Design: We used quantitative real-time PCR to measure TLR expression in the decidua and DSCs (decidual stromal cells). DSCs were cultured in the presence or absence of the TLR2 agonists PAM3CSK4, PGN-Sa, and zymosan, the TLR3 agonist poly (I:C) and the TLR4 agonist LPS. Then, the supernatants were assayed for CCL2 secreted by DSCs and IL-4, IFN-γ, IL-10, and TNF-α produced by DICs., Results: Costimulation with TLR2, TLR3 and TLR4 agonists resulted in enhancing CCL2 production compared with that in the controls. Additionally, these TLR2, 3, and 4 agonists stimulated CD80/CD86 on DSCs and regulated IL-4 and IL-10 secretion on DICs. TLR2 and TLR3 agonists may promote Th1/Th2 immune bias., Conclusions: TLRs may induce Th1/Th2 responses by affecting the secretion of CCL2 at the maternal-foetal interface., (Copyright © 2021. Published by Elsevier B.V.)

Tumor-derived exosomes (TEXs) could be harnessed as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). Engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic., (Copyright © 2021 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility., Competing Interests: The authors have declared that no competing interests exist.

In this study, we report that the TLR4 ligand, LPS, and TLR3 ligand polyinosinic:polycytidylic acid failed to activate IRF3 or STAT1 in bone marrow-derived macrophages (BMMs) isolated from two independently generated lines of Rosa26-integrated Cas9-expressing C57BL/6J (B6) mice. RNA-sequencing analysis reveals that hundreds to thousands of genes including IFN-stimulated genes were differentially expressed in BMMs from these Cas9 strains compared with B6 upon LPS stimulation. Furthermore, the NF-κB signaling axis and TRIF-mediated necroptosis were also strongly reduced in response to LPS and polyinosinic:polycytidylic acid. In contrast, there were no defects in the responses of BMMs to ligands of the RIG-I, STING, TLR2, TLR9, and IFN receptors. Defects in TLR3 and TLR4 signaling were observed in mice with the B6 but not 129 background, and when Cas9 was integrated at the Rosa26 but not H11 locus. However, integration at the Rosa26 site, CAG promoter-driven Cas9 or eGFP were not individually sufficient to cause the defect. Taken together, the results of this study suggest a putative TRIF-mediated defect in TLR-3/4 signaling in BMMs from commercially available and widely used B6-Cas9-expressing mice., (Copyright © 2021 The Authors.)

The interferon receptor system in teleost fish is more complex than that in mammals. In the present study, we identified 13 cytokine receptor genes (10 interferon receptor genes and 3 IL10R2-like genes) from Chinese sturgeon (Acipenser sinensis) using RNA-sequencing. Sequence analysis indicated that these receptors had conserved domains, including signal peptides, FNⅢ, and transmembrane domains. Phylogenetic analysis suggested that they belonged to the cytokine receptor family. In the present study, we named them IFNAR1-like (CRFB5a, CRFB5b), IFNAR2-like (CRFB3a, CRFB3b), IFNGR1-like (IFNGR1), IFNGR2-like (CRFB6a, CRFB6b/IFNGR2-1, CRFB6c/IFNGR2-2, CRFB6d/IFNGR2-3, CRFB6e/IFNGR2-4) and IL10R2-like (CRFB4a, CRFB4b, CRFB4c), respectively. Constitutive expression analysis revealed that these receptor genes had potential functions in immune and non-immune tissue compartments. After stimulating with Poly (I:C), the expression fold changes of CRFB3a, CRFB4a, CRFB4b, CRFB5b, and CRFB6e/IFNGR2-4 in Chinese sturgeon were higher than those of other receptor genes, which revealed that these five genes had important functions in the immune process to resist virus invasion in the host. After stimulating with IFN gamma, the expression fold changes of CRFB3a, CRFB4a, and CRFB6b/IFNGR2-1 were higher than those other receptor genes. Based on other teleost fish interferon receptor models, we speculated that IFNAR1-like (CRFB5a, CRFB5b) and IFNAR2-like (CRFB3a, CRFB3b), comprised Chinese sturgeon type Ⅰ IFN receptors; and IFNGR1-like (IFNGR1) and IFNGR2-like (CRFB6/IFNGR2) comprised Chinese sturgeon type Ⅱ IFN receptors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Toll-interacting protein (Tollip) and MyD88 are key components of the TLR/IL-1R signaling pathway in mammals. MyD88 is known as a universal adaptor protein involving in TLR/IL-1R-induced NF-κB activation. Tollip is a crucial negative regulator of TLR-mediated innate immune responses. Previous studies have demonstrated that teleost Tollip served as a negative regulator of MyD88-dependent TLR signaling pathway. However, the mechanism is still unclear. In particular, the effect of TBD, C2, and CUE domains of Tollip on MyD88-NF-κB signaling pathway remains to be elucidated. In this study, we found that the response of grass carp Tollip (CiTollip) to LPS stimulation was faster and stronger than that of poly I:C treatment, and CiTollip diminished the expression of tnf-α induced by LPS. Further assays indicated that except for the truncated mutant of △CUE2 (1-173 aa), wild type CiTollip and other truncated mutants (△N-(52-276 aa), △C2-(173-276 aa) and △CUE1-(1-231 aa)) could associate with MyD88 and negatively regulate MyD88-induced NF-κB activation. It suggested that the C-terminal (173-276 aa), in particular the connection section between C2 and CUE domains (173-231 aa), played a pivotal role in suppressing MyD88-induced activation of NF-κB., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named CgTRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgTRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted CgTRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the CgTRIM1 was classified as the member of C-V TRIM subfamily. The mRNA transcripts of CgTRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of CgTRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of CgTRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (CgIFNLP) was knocked down by RNA interference (RNAi), the expression of CgTRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of CgTRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (CgMx) and Interferon-induced protein 44 (CgIFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the CgIFNLP expression was observed. These results indicated that CgTRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mosquito anti-pathogen immune responses, including those controlling infection with arboviruses are regulated by multiple signal transduction pathways. While the Toll pathway is critical in the defense against arboviruses such as dengue and Zika viruses, the factors and mechanisms involved in virus recognition leading to the activation of the Toll pathway are not fully understood. In this study we evaluated the role of virus-produced double-stranded RNA (dsRNA) intermediates in mosquito immune activation by utilizing the synthetic dsRNA analog polyinosinic-polycytidylic acid (poly I:C). Poly I:C treatment of Aedes aegypti mosquitoes and Aag2 cells reduced DENV infection. Transcriptomic analyses of Aag2 cell responses to poly I:C indicated putative activation of the Toll pathway. We found that poly I:C is translocated to the endosomal compartment of Aag2 cells, and that the A. aegypti Toll 6 receptor is a putative dsRNA recognition receptor. This study elucidates the role of dsRNAs in the immune activation of non-RNAi pathways in mosquitoes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Toll/interleukin-1 receptor (TIR) domain-containing adaptors, serve as pivotal signal transduction molecules in Toll-like receptor (TLR) signalling pathway to mediate downstream signalling cascades. In this study, four TIR-domain containing adaptors, MyD88, TRIF, MAL and SARM, were identified in mandarin fish Siniperca chuatsi, and they all contain TIR domains, of which MyD88 and SARM had high sequence homology with their vertebrate homologues. The expression analysis at mRNA level indicated that these genes were ubiquitously distributed in different tissues, being high in immune- and mucosa-related tissues such as head-kidney and intestine. The transcripts of these adaptor genes were up-regulated by poly(I:C) and LPS stimulation in isolated head-kidney lymphocytes (HKLs) of mandarin fish. Fluorescence microscopy revealed that all these molecules were localized in cytoplasm, and further investigations showed that the over-expression of MyD88, TRIF and MAL activated the NF-κB, ISRE or type Ι IFN promoters and inhibited SVCV replication, whereas their antiviral effects were significantly impaired when co-transfected with SARM. It was also confirmed by co-immunoprecipitation (Co-IP) that SARM interacts separately with MyD88, TRIF and MAL, and MAL interacts with MyD88. However, the regulatory mechanisms of these adaptors involved in signalling pathways of different TLRs should be of interest for further research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mammalian Nemo-like kinase (NLK) plays important roles in multiple biological processes including immune response; however, the roles of teleost NLK remain largely unknown. In the present study, the NLK homolog (bcNLK) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The coding region of bcNLK consists of 1427 nucleotides and encodes 476 amino acid, including two low complexity region (LCR) domains at the N-terminus and a serine/threonine protein kinase catalytic (S-TKc) domain in the middle region. The transcription of bcNLK are promoted after spring viremia of carp virus (SVCV) infection and poly (I:C) stimulation in host cells, but not post LPS treatment. bcNLK exhibits weak impact on the transcription of interferon (IFN) promoter in the reporter assay, however, black carp MAVS (bcMAVS)-mediated IFN promoter transcription is remarkably dampened by bcNLK. The interaction between bcNLK and bcMAVS is detected through the co-immunoprecipitation assay. Accordingly, the plaque assay results show that bcMAVS-mediated antiviral ability is impaired by bcNLK. Moreover, knockdown of bcNLK in host cells leads to the enhanced antiviral ability against SVCV. All these data support the conclusion that black carp NLK associates with MAVS and inhibited MAVS-mediated antiviral signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1β, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

CD226 interacts with its ligand Necl5 as a costimulatory signal. In this study, we cloned a CD226 from Nile tilapia (Oreochromis niloticus, named OnCD226) and a Necl5 (named OnNecl5). The open reading frame of OnCD226 was 1071 bp, encoding a protein of 356 amino acids. Sequence alignment analysis indicated that OnCD226 contained two Ig-like domains in ectodomain. The open reading frame of OnNecl5 was 1155 bp, encoding a protein of 384 amino acids, and there are three lg-like domains in the extracellular domain. In healthy tilapia, OnCD226 was distributed in all tested tissues and relatively higher in the brain, while OnNecl5 was relatively higher in the skin. After Streptococcus agalactiae infection, OnCD226 has the same up-regulated expression pattern as OnNecl5 in different tissues. After HKLs stimulation with S. agalactiae and Poly I:C, respectively. OnCD226 was significantly up-regulated (0.01 < p < 0.05) at 12 h and extremely significant up-regulation was observed (p < 0.01) at 48 h and 96 h, the peak was observed at 96 h after stimulation by S. agalactiae. After stimulation by Poly I:C, OnCD226 expression was extremely significant (p < 0.01) at 72 h and 96 h, the peak was observed at 96 h. After stimulation by Keyhole limpet hemocyanin (KLH), a classical T cell-dependent antigen, the expression of OnCD226 was significantly up-regulated in blood, head kidney, spleen, and thymus. Moreover, when compared with the first challenge, the gene expression of OnCD226 which response to the second challenge was up-regulated earlier. Subcellular co-localization studies showed that OnCD226 and OnNecl5 were distributed mainly in the cytomembrane. Yeast two-hybrid results, indicated a strong interaction between OnCD226 and OnNecl5. These results suggested that OnCD226 plays an important role during pathogens infection, and the interaction between CD226 and Necl5 is conserved in Nile tilapia., (Copyright © 2021. Published by Elsevier Ltd.)

Toll-like receptors (TLRs), as a family of pattern recognition receptors (PRRs), possess specific pathogen-related molecular pattern (PAMP) recognition spectrum in inducing immune responses. In this study, sixteen TLRs were identified and characterized in mandarin fish (Siniperca chuatsi). All these TLRs consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor (TIR) domain, with the exception of TLR5S which lacks TIR domain, and they can be clustered into five branches, i.e. TLR1 subfamily, TLR3 subfamily, TLR5 subfamily, TLR7 subfamily and TLR11 subfamily in phylogenetic tree. These TLR genes were expressed in all tested tissues and had high expression levels in immune-related tissues such as head-kidney and spleen or mucosa-related tissues such as intestine and pyloric caecum. The transcripts of TLR2a, TLR2b, TLR3, TLR13a, TLR14, TLR22 and TLR23 were all significantly up-regulated after stimulation with poly(I:C); TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR13a and TLR13b transcripts were all significantly up-regulated after stimulation with PGN; and TLR2a, TLR2b, TLR5M, TLR5S, TLR7, TLR8, TLR9, TLR13c, TLR14 and TLR22 transcripts were all significantly up-regulated after stimulation with LPS in isolated head kidney lymphocytes of mandarin fish. The findings in this study may provide a valuable basis for functional study on TLR genes in mandarin fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transcription factor small mothers against decapentaplegic (Smad) family SMAD proteins are the essential intracellular signal mediators and transcription factors for transforming growth factor β (TGF-β) signal transduction pathway, which usually exert pleiotropic actions on cell physiology, including immune response, cell migration and differentiation. In this study, the Smad family was identified in the most primitive vertebrates through the investigation of the transcriptome data of lampreys. The topology of phylogenetic tree showed that the four Smads (Smad1, Smad3, Smad4 and Smad6) in lampreys were subdivided into four different groups. Meanwhile, homology analysis indicated that most Smads were conserved with typical Mad Homology (MH) 1 and MH2 domains. In addition, Lethenteron reissneri Smads (Lr-Smads) adopted general Smads folding structure and had high tertiary structural similarity with human Smads (H-Smads). Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey Smads presented dramatic differences compared with jawed vertebrates. Importantly, quantitative real-time PCR analysis demonstrated that Smads were widely expressed in lamprey, and the expression level of Lr-Smads mRNA was up-regulated with different pathogenic stimulations. Moreover, depending on the weighted gene co-expression network analysis (WGCNA), four Lr-Smads were identified as two meaningful modules (green and gray). The functional analysis of these two modules showed that they might have a correlation with ployI:C. And these genes presented strong positive correlation during the immune response from the results of Pearson's correlation analysis. In conclusion, our results would not only enrich the information of Smad family in jawless vertebrates, but also lay the foundation for immunity in further study., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mitochondrial antiviral signaling protein (MAVS) acts as an essential adaptor in host RIG-I-like receptors (RLRs) mediated antiviral signaling pathway. In the present study, two MAVS transcript variants, the typical form and a splicing variant, namely Lc-MAVS&#95;tv1 and Lc-MAVS&#95;tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-MAVS&#95;tv1 protein contains 512 aa, with an N-terminal CARD domain, a central proline-rich region, and a C-terminal transmembrane (TM) domain, whereas Lc-MAVS&#95;tv2 contains 302 aa and lacks the C-terminal TM domain due to a premature stop in the 102 bp intron fragment insertion. Lc-MAVS&#95;tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS&#95;tv2 exhibited an entire cytosolic distribution. Quantitative real-time PCR revealed that Lc-MAVS&#95;tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-MAVS&#95;tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression of Lc-MAVS&#95;tv2 could induce the activation of NF-κB but not IRF3, and Lc-MAVS&#95;tv2 co-transfected with Lc-MAVS&#95;tv1 induced a significantly higher level of NF-κB and IRF3 promoter activity. In addition, Lc-MAVS&#95;tv2 overexpression could enhance TRAF3 and TRAF6 mediated NF-κB activation, but suppress TRAF3 and TRAF6 mediated IRF3 activation, implying that the splicing variant Lc-MAVS&#95;tv2 may function as an important regulator in MAVS mediated signaling pathway., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Type I IFNs (IFN-I) are important for tumor immune surveillance and contribute to the therapeutic responses for numerous treatment regimens. Nevertheless, certain protumoral activities by IFN-I have been increasingly recognized. Indeed, our recent work showed that systemic poly(I:C)/IFN treatment can undesirably trigger high arginase (ARG1) expression within the tumor-associated monocyte/macrophage compartment. Using a line of CRISPR-generated Arg1-YFP reporter knock-in mice, we have determined that a subset of tumor-associated macrophages represent the major Arg1 -expressing cell type following poly(I:C)/IFN stimulation. More detailed analyses from in vitro and in vivo models demonstrate a surprising IFN-to-IL-4 cytokine axis in transitional monocytes, which can subsequently stimulate IL-4 target genes, including Arg1 , in macrophages. Intriguingly, IFN stimulation of transitional monocytes yielded concurrent M2 (YFP + )- and M1 (YFP - )-skewed macrophage subsets, correlated with an inhibitory crosstalk between IFN-I and IL-4. Genetic abrogation of IL-4 signaling in mice diminished poly(I:C)/IFN-induced ARG1 in tumors, leading to enhanced activation of CD8 + T cells and an improved therapeutic effect. The present work uncovered a monocyte-orchestrated macrophage phenotype conversion mechanism that may have broad implications., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Problem: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes., Method of Study: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality., Results: Our findings demonstrated a significant induction of testicular TNF-α, IL-1β and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier., Conclusion: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-β expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-β, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-β, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Indo, Kitahara, Tomokiyo, Araki, Islam, Zhou, Albarracin, Miyazaki, Ikeda-Ohtsubo, Nochi, Takenouchi, Uenishi, Aso, Takahashi, Kurata, Villena and Kitazawa.)

Background: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs)., Method: Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex., Results: Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients., Conclusion: MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.