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PA-X protein arises from a ribosomal frameshift in the PA of influenza A virus (IAV). However, the immune regulatory effect of the PA-X protein of H1N1 viruses on the nasal mucosal system remains unclear. Here, a PA-X deficient H1N1 rPR8 viral strain (rPR8-△PAX) was generated and its pathogenicity was determined. The results showed that PA-X was a pro-virulence factor in mice. Furthermore, it reduced the ability of H1N1 viruses to infect dendritic cells (DCs), the regulator of the mucosal immune system, but not non-immune cells (DF-1 and Calu-3). Following intranasal infection of mice, CCL20, a chemokine that monitors the recruitment of submucosal DCs, was downregulated by PA-X, resulting in an inhibition of the recruitment of CD11b+ DCs to submucosa. It also attenuated the migration of CCR7+ DCs to cervical lymph nodes and inhibited DC maturation with low MHC II and CD40 expression. Moreover, PA-X suppressed the maturation of phenotypic markers (CD80, CD86, CD40, and MHC II) and the levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhancing endocytosis and levels of anti-inflammatory IL-10 in vitro, suggesting an impaired maturation of DCs that the key step for the activation of downstream immune responses. These findings suggested that the PA-X protein played a critical role in escaping the immune response of nasal mucosal DCs for increasing the virulence of H1N1 viruses.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

Abstract Influenza virus with numerous subtypes and frequent variation limits the development of high‐efficacy and broad‐spectrum antiviral strategy. Here, a novel multi‐antiviral metastable iron sulfides (mFeS) against various influenza A/B subtype viruses is developed. This work finds that mFeS induces high levels of lipid peroxidation and •OH free radicals in the conservative viral envelope, which depends on Fe2+. This phenomenon, termed as a viral ferroptosis, results in the loss of viral infectibility and pathogenicity in vitro and in vivo, respectively. Furthermore, the decoction of mFeS (Dc(mFeS)) inhibits cellular ferroptosis‐dependent intracellular viral replication by correcting the virus‐induced reprogrammed sulfur metabolism, a conserved cellular metabolism. Notably, personal protective equipment (PPE) that is loaded with mFeS provides good antiviral protection. Aerosol administration of mFeS combined with the decoction (mFeS&Dc) has a potential therapeutic effect against H1N1 lethal infection in mice. Collectively, mFeS represents an antiviral alternative with broad‐spectrum activity against intracellular and extracellular influenza virus.

ABSTRACT: H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

In the bacterial infection of humans, sepsis is of great concern based on its high fatality rate. Correction of immune dysfunction by targeting dendritic cells (DCs) is a promising strategy for sepsis therapy. Royal jelly, a natural health product, is produced by worker bees for feeding queens. In the study, we evaluated whether royal jelly could attenuate LPS-induced immune dysfunction of DCs. We found that royal jelly reduced the expression levels of mature phenotype (MHCII, CD40, CD80, and CD86), activation marker (CD69), and inflammatory cytokines (TNF-α, IL-6, and IL-10) in LPS-induced DCs. In addition, royal jelly enhanced the endocytosis levels and abrogated allogeneic CD4+ T cell proliferation in LPS-induced DCs. Finally, we found that the Nrf2/HO-1 axis played a vital role in the inhibition of LPS-induced immune dysfunction of DCs by royal jelly. These results indicated that royal jelly had a potential application in sepsis therapy by regulating DC function.

Holarrhena pubescens Wall. ex G. Don (H. pubescens), belonging to the Apocynaceae family, is distributed in deciduous forests of the tropical Himalayas. H. pubescens is an important traditional medicinal plant, especially its seeds and barks. Therefore, we assessed the antioxidant capacity of H. pubescens extracts in Lipopolysaccharide (LPS)-induced dendritic cells (DCs) for sepsis treatment. Our results indicated that H. pubescens extracts with different doses (25 μg/mL, 50 μg/mL, 100 μg/mL) reduced the reactive oxygen species (ROS) level, and weakened the nitric oxide synthases (NOS) activity and nitric oxide (NO) level in LPS (100 ng/mL)-irritated DCs. In addition, H. pubescens extracts decreased the oxidized glutathione (GSSG) production but increased the reduced glutathione (GSH) production, thereby preserving the cellular reductive status owing to the raised GSH/GSSG ratio. Furthermore, H. pubescens extracts strengthened the antioxidant enzymes activity in LPS-induced DCs, such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). Finally, we found that H. pubescens extracts significantly improved the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the heme oxygenase 1 (HO–1) in LPS-irritated DCs. These results indicated that H. pubescens extracts suppressed the LPS-irritated oxidative stress in DCs via Nrf2/HO–1 signaling pathway, providing a potential strategy for sepsis therapy.

Melatonin, an indoleamine synthesized in the pineal gland of mammals, is a natural bioactive compound with powerful antioxidant and anti-inflammatory properties. Here, we evaluated whether melatonin has the capacity to moderate the oxidative stress of dendritic cells (DCs) for inflammatory control in an acute lung injury (ALI) model. Our findings showed that melatonin remarkably inhibited total nitric oxide synthase (T-NOS) activity, nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, and lipid peroxidation (MDA detection) levels in both an LPS-induced murine ALI model and LPS-induced DCs. Meanwhile, the reduced glutathione (GSH) level and the GSH/GSSG ratio were recovered. In addition, antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were increased in these processes. Moreover, melatonin also inhibited the LPS-induced secretions of IL-1β, IL-6, and TGF-β in vivo and in vitro. Finally, we found that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) axis was required in the inhibition of LPS-induced oxidative stress in DCs by melatonin. Altogether, these data indicate that melatonin strongly suppresses the LPS-induced oxidative stress in DCs, which is a promising DC-targeted strategy via inflammatory control for ALI treatment.

Abstract Influenza poses a severe threat to global health. Despite the whole inactivated virus (WIV)‐based nasal vaccine being a promising strategy for influenza protection, the mucosal barrier is still a bottleneck of the nasal vaccine. Here, a catalytic mucosal adjuvant strategy for an influenza WIV nasal vaccine based on chitosan (CS) functionalized iron oxide nanozyme (IONzyme) is developed. The results reveal that CS‐IONzyme increases antigen adhesion to nasal mucosa by 30‐fold compared to H1N1 WIV alone. Next, CS‐IONzyme facilitates H1N1 WIV to enhance CCL20‐driven submucosal dendritic cell (DC) recruitment and transepithelial dendrite(TED) formation for viral uptake via the toll‐like receptor(TLR) 2/4‐dependent pathway. Moreover, IONzyme with enhanced peroxidase (POD)‐like activity by CS modification catalyzes a reactive oxygen species (ROS)‐dependent DC maturation, which further enhances the migration of H1N1 WIV‐loaded DCs into the draining lymph nodes for antigen presentation. Finally, CS‐IONzyme‐based nasal vaccine triggers an 8.9‐fold increase of IgA‐mucosal adaptive immunity in mice, which provides a 100% protection against influenza, while only a 30% protection by H1N1 WIV alone. This work provides an antiviral alternative for designing nasal vaccines based on IONzyme to combat influenza infection.

Intranasal immunization with whole inactivated virus (WIV) is an important strategy used for influenza prevention and control. However, a powerful mucosal adjuvant is required to improve nasal vaccine efficacy. Riboflavin, as a food additive with the advantages of being safe and low-cost, widely exists in living organisms. In this paper, the mucosal adjuvant function of riboflavin was studied. After intranasal immunization with H1N1 WIV plus riboflavin in mice, we found that the mucosal immunity based on the secretory IgA (sIgA) levels in the nasal cavity, trachea, and lung were strongly enhanced compared with H1N1 WIV alone. Meanwhile, the IgG, IgG1, and IgG2a levels in serum also showed a high upregulation and a decreased ratio of IgG1/IgG2a, which implied a bias in the cellular immune response. Moreover, riboflavin strongly improved the protection level of H1N1 inactivated vaccine from a lethal influenza challenge. Furthermore, riboflavin was found to possess the capacity to induce dendritic cell (DC) phenotypic (MHCII, CD40, CD80, and CD86) and functional maturation, including cytokine secretion (TNF-α, IL-1β, IL-12p70, and IL-10) and the proliferation of allogeneic T cells. Lastly, we found that the DC maturation induced by riboflavin was dependent on the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which plays an important role in immune regulation. Therefore, riboflavin is expected to be developed as an alternative mucosal adjuvant for influenza nasal vaccine application.

Astaxanthin, originating from marine organisms, is a natural bioactive compound with powerful antioxidant activity. Here, we evaluated the antioxidant ability of astaxanthin on dendritic cells (DCs), a key target of immune regulation, for inflammatory control in a sepsis model. Our results showed that astaxanthin suppressed nitric oxide (NO) production, reactive oxygen species (ROS) production, and lipid peroxidation activities in LPS-induced DCs and LPS-challenged mice. Moreover, the reduced glutathione (GSH) levels and the GSH/GSSG ratio were increased, suggesting that astaxanthin elevated the level of cellular reductive status. Meanwhile, the activities of antioxidant enzymes, including glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were significantly upregulated. Astaxanthin also inhibited the LPS-induced secretions of IL-1β, IL-17, and TGF-β cytokines. Finally, we found that the expressions of heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly upregulated by astaxanthin in LPS-induced DCs, suggesting that the HO-1/Nrf2 pathway plays a significant role in the suppression of oxidative stress. These results suggested that astaxanthin possesses strong antioxidant characteristics in DC-related inflammatory responses, which is expected to have potential as a method of sepsis treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) has received increasing attention in recent years. However, the characteristics and relevant mechanisms of biofilm formation in oxacillin-sensitive MRSA (OS-MRSA) are poorly understood. This study was designed to characterize biofilm formation in OS-MRSA BWSA15 in response to ceftazidime (TZ) by comparing the methicillin-sensitive S. aureus (MSSA) strain BWSA23 and the oxacillin-resistant MRSA (OR-MRSA) strain BWSA11. The biofilms and biofilm-forming cells were observed by electron microscopy. Biofilms grown on microtiter plates were chemically decomposed and analyzed by Fourier transform infrared spectroscopy. The transcriptional regulation of genes associated with methicillin resistance, surface adhesion, fatty acid biosynthesis, and global regulation (sigma B) was investigated. A significant increase in biofilm formation ability (10.21-fold) and aggregation ability (2.56-fold) was observed in BWSA15 upon the treatment with TZ (16 μg/ml). The TZ-induced biofilm formation in BWSA15 was characterized by a disappearance of polysaccharide-like extracellular substances and an appearance of a large number of intercellular MVs from extracellular matrix. Few MVs were identified in the biofilms formed by BWSA11 and BWSA23. There was a significant upregulation of mecA, sigB, and fatty acid biosynthesis-associated genes and downregulation of icaA, icaD, clfA, clfB, and fnaA in BWSA15 upon the treatment with TZ. The formation of intracellular junctions of MVs in the biofilms of BWSA15 was mediated by a significant increase in the proportion of proteins as well as by an increase in the proportion of non-ionized carboxyl groups in fatty acids. This study demonstrated that beta-lactam antibiotics can induce biofilm formation in OS-MRSA, and the biofilm induction in OS-MRSA can mainly be attributed to exposed MVs with increased hydrophobicity rather than polysaccharide intercellular adhesins, cell wall-anchored surface proteins, and extracellular DNA.

Astaxanthin, originating from seafood, is a naturally occurring red carotenoid pigment. Previous studies have focused on its antioxidant properties; however, whether astaxanthin possesses a desired anti-inflammatory characteristic to regulate the dendritic cells (DCs) for sepsis therapy remains unknown. Here, we explored the effects of astaxanthin on the immune functions of murine DCs. Our results showed that astaxanthin reduced the expressions of LPS-induced inflammatory cytokines (TNF-α, IL-6, and IL-10) and phenotypic markers (MHCII, CD40, CD80, and CD86) by DCs. Moreover, astaxanthin promoted the endocytosis levels in LPS-treated DCs, and hindered the LPS-induced migration of DCs via downregulating CCR7 expression, and then abrogated allogeneic T cell proliferation. Furthermore, we found that astaxanthin inhibited the immune dysfunction of DCs induced by LPS via the activation of the HO-1/Nrf2 axis. Finally, astaxanthin with oral administration remarkably enhanced the survival rate of LPS-challenged mice. These data showed a new approach of astaxanthin for potential sepsis treatment through avoiding the immune dysfunction of DCs.

Dendritic cells (DCs) play a vital role in the regulation of immune-mediated inflammatory diseases. Thus, DCs have been regarded as a major target for the development of immunomodulators. However, oxidative stress could disturb inflammatory regulation in DCs. Here, we examined the effect of bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, on the protection of DCs against oxidative stress for immunosuppression. BP5 showed potent protective effects against the lipopolysaccharide (LPS)-induced oxidative stress in DCs, including nitric oxide, reactive oxygen species and lipid peroxidation. Furthermore, BP5 elevated the level of cellular reductive status through increasing the reduced glutathione (GSH) and the GSH/GSSG ratio. Concomitant with these, the activities of several antioxidative redox enzymes, including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), were obviously enhanced. BP5 also suppressed submucosal DC maturation in the LPS-stimulated intestinal epithelial cells (ECs)/DCs coculture system. Finally, we found that heme oxygenase 1 (HO-1) was remarkably upregulated by BP5 in the LPS-induced DCs, and played an important role in the suppression of oxidative stress and DC maturation. These results suggested that BP5 could protect the LPS-activated DCs against oxidative stress and have potential applications in DC-related inflammatory responses.

Context. The immune system of piglets is not fully developed at weaning. The immature adaptive immune system along with the change in the feed types and the living conditions make piglets susceptible to pathogenic infections, and result in diarrhoea and decreased growth. However, the underlying mechanisms require further research. Aims. This study was conducted to investigate the effects of weaning on the growth performance and intestinal immune function of piglets. Methods. In total, 40 piglets with similar bodyweights were selected in pairs at 21 days old and divided into the suckling group (SG, breastfed by their mothers) and weaning group (WG, weaned at 21 days old). Eight piglets from each group were randomly selected and sacrificed at 24 days (SG3 and WG3) and 28 days of age (SG7 and WG7). The growth performance, T lymphocyte subpopulations, the concentration of cytokines and immunoglobulins, and the expression of Notch2 signalling proteins were determined. Key results. The weaning caused a decrease in bodyweight (P < 0.01) and the ratio of CD3+CD4+/CD3+CD8+ T cells in the thymus (P < 0.05). Compared with SG3, the concentration of secretory immunoglobulin A (sIgA) in the jejunum was decreased, and that of interleukin 2 (IL-2) in serum and ileum, IL-1β and IL-2 in jejunum were upregulated (P < 0.01), while IL-10 in the small intestine was downregulated (P < 0.05) in WG3. Weaning downregulated gene expression of IL-4 and upregulated gene expression of IL-1β, IL-12, and interferon γ (IFN-γ) in small intestine (P < 0.05). Weaning downregulated protein expression of Notch2 and Hes1 but upregulated Jagged1 expression in small intestine of piglets (P < 0.05). Conclusions. Weaning caused an imbalance in T lymphocyte distribution, thus impairing the intestinal immune function of piglets, which might be associated with the Notch2 signalling. Furthermore, weaning caused adverse effects on piglets especially on the first 3 days, which were partly recovered after 3 days, but still existed at 7 days after weaning. Implications. The results of this study may guide the pig-raising industry, especially for the management of piglets shortly after weaning. [ABSTRACT FROM AUTHOR]

The spread of the low-pathogenicity avian H9N2 influenza virus has seriously increased the risk of a new influenza pandemic. Although whole inactivated virus (WIV) vaccine via intranasal pathway is the effective method of blocking virus transmission, the mucosal barrier seems to be a major factor hampering its development. CpG oligodeoxynucleotides, a known adjuvant, can target downstream dendritic cells (DCs) and effectively enhance the mucosal and systemic immune responses. However, the ability of CpGs to assist H9N2 WIV in transepithelial transport remains unknown. Here, in vitro and in vivo, we showed that CpGs provided assistance for H9N2 WIV in recruiting DCs to the nasal epithelial cells (ECs) and forming transepithelial dendrites (TEDs) to capture luminal viruses. CD103+ DCs participated in this process. Chemokine CCL20 from nasal ECs played a key role in driving DC recruitment and TED formation. Virus-loaded DCs quickly migrated into the draining cervical lymph nodes (CLNs) for antigen presentation. In addition, the competence of CpGs was independent of direct epithelial transport via the transcellular or paracellular pathway. Taken together, our data demonstrated that CpGs enhanced the transport of H9N2 WIV via TEDs of nasal DCs, which might be a novel mechanism for optimal adaptive immune responses. [ABSTRACT FROM AUTHOR]