yan Yin

Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella-induced apoptosis in Caco-2 cells.

Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits present as the outermost component of the cell wall in several Lactobacillus species. The underlying mechanism for how S-layer proteins inhibit pathogen infections remains unclear. To gain insights into the mechanism of the antimicrobial activity of Lactobacillus S-layer proteins, we examined how Lactobacillus S-layer proteins impact Salmonella Typhimurium-induced apoptosis in vitro in Caco-2 human colon epithelial cells. When Caco-2 cells infected with Salmonella Typhimurium SL1344, we found that apoptosis was mediated by activation of caspase-3, but not caspase-1. When Salmonella Typhimurium SL1344 and S-layer proteins were coincubated simultaneously, Caco-2 cell apoptosis was markedly decreased and the cell damage was modified, as evaluated by flow cytometry and microscopy. Detailed analyses showed that the S-layer proteins inhibited the caspase-3 activity and activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway. Taken together, these findings suggest that Lactobacillus S-layer proteins protected against Salmonella-induced apoptosis through reduced caspase-3 activation. In addition, Salmonella-induced apoptotic cell damage was modified by S-layer proteins through the ERK1/2 signaling pathway. This mechanism may represent a novel approach for antagonizing Salmonella infection.

Chicken bone marrow-derived dendritic cells maturation in response to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is highly contagious disease which easily lead to immunosuppression and a decreased response to vaccinations in young chicken. Since dendritic cells (DCs) are crucial to induce immunity and their maturation and functions are influenced by microbial and environmental stimuli, we investigated the effects of inactivated IBDV and IBDV on chicken DC activation and maturation. Chicken bone marrow-derived dendritic cells (chBM-DCs) cultured in complete medium (including recombinant chicken: granulocyte-macrophage colony-stimulating factor and interleukin 4) expressed high levels of MHC-II and the putative CD11c. After LPS or virus stimulation, chBM-DCs displayed the typical morphology of DCs. In addition, stimulation by LPS or viruses significantly elevated chBM-DCs surface expression levels of CD40 and CD86 molecules, as well as the ability to induce T-cell proliferative response, compared to the non-stimulated chBM-DCs. Interestingly, inactive IBDV showed stronger ability to up-regulate expression levels of CD40 and CD86 molecules and stimulate naive T cells proliferation than live IBDV. These results revealed that live viruses infection impaired DC maturation and functions, probably explaining why chickens infected with IBDV fails to trigger an effective specific immune response or develop immune memory.

H9N2 Influenza Whole Inactivated Virus Combined with Polyethyleneimine Strongly Enhances Mucosal and Systemic Immunity after Intranasal Immunization in Mice

ABSTRACT Influenza whole inactivated virus (WIV) is more immunogenic and induces protective antibody responses compared with other formulations, like split virus or subunit vaccines, after intranasal mucosal delivery. Polyethyleneimine (PEI), an organic polycation, is widely used as a reagent for gene transfection and DNA vaccine delivery. Although PEI recently has demonstrated potent mucosal adjuvant activity for viral subunit glycoprotein antigens, its immune activity with H9N2 WIV is not well demonstrated. Here, mice were immunized intranasally with H9N2 WIV combined with PEI, and the levels of local respiratory tract and systemic immune responses were measured. Compared to H9N2 WIV alone, antigen-specific IgA levels in the local nasal cavity, trachea, and lung, as well as levels of IgG and its subtypes (IgG1 and IgG2a) in the serum, were strongly enhanced with the combination. Similarly, the activation and proliferation of splenocytes were markedly increased. In addition, PEI is superior as an H9N2 WIV delivery system due to its ability to greatly increase the viral adhesion to mucosal epithelial cells and to enhance the cellular uptake and endosomal escape of antigens in dendritic cells (DCs) and further significantly activate DCs to mature. Taken together, these results provided more insights that PEI has potential as an adjuvant for H9N2 particle antigen intranasal vaccination.

Bursopentin (BP5) from chicken bursa of fabricius attenuates the immune function of dendritic cells.

Bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, has been proved to have immunomodulatory effects on B and T lymphocytes, anti-oxidative stress on macrophages, and antiproliferation on tumor cells. However, the effects of BP5 on the immune function exhibited by dendritic cells (DCs), which are regarded as a major target for immunomodulators, remain unknown. In this study, we examined the effects of BP5 on the activation and maturation of murine bone marrow-derived DCs. Our results showed that BP5 significantly suppressed the secretion of lipopolysaccharide (LPS)-induced pro-inflammatory (TNF-α, IL-1β, IL-6 and IL-12p70) and anti-inflammatory (IL-10) cytokines by DCs, and this impact was not due to its cytotoxicity. Besides, BP5 reversed the morphological changes and attenuated the expression of phenotypic markers (MHC II, CD40, CD80 and CD86 molecules) in LPS-induced DCs. Furthermore, BP5 restored the decreased FITC-dextran uptake in LPS-treated DCs, arrested the LPS-induced migration of DCs and abrogated the promoting ability of LPS-induced DCs for allogeneic T cell proliferation. These findings show a new immunopharmacological capability of BP5 and provide a novel approach in the prevention and therapy of chronic inflammation and autoimmunity via abolishing the immune function of DCs.

CpG DNA assists the whole inactivated H9N2 influenza virus in crossing the intestinal epithelial barriers via transepithelial uptake of dendritic cell dendrites.

Intestinal mucosa remains a pivotal barrier for the oral vaccine absorption of H9N2 whole inactivated influenza virus (WIV). However, CpG DNA, as an adjuvant, can effectively improve relevant mucosal and systemic immunity. The downstream mechanism is well confirmed, yet the evidence of CpG DNA assisting H9N2 WIV in transepithelial delivery is lacking. Here, we reported both in vitro and in vivo that CpG DNA combined with H9N2 WIV was capable of recruiting additional dendritic cells (DCs) to the intestinal epithelial cells (ECs) to form transepithelial dendrites (TEDs) for luminal viral uptake. Both CD103+ and CD103− DCs participated in this process. The engagement of the chemokine CCL20 from the apical ECs and the DCs drove DC recruitment and TED formation. Virus-loaded CD103+ but not CD103− DCs also quickly migrated into mesenteric lymph nodes within 2 h. Moreover, the mechanism of CpG DNA was independent of epithelial transcytosis and disruption of the epithelial barriers. Finally, the subsequent phenotypic and functional maturation of DCs was also enhanced. Our findings indicated that CpG DNA improved the delivery of H9N2 WIV via TEDs of intestinal DCs, and this may be an important mechanism for downstream effective antigen-specific immune responses.

Effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen.

Abstract Virulent transmissible gastroenteritis virus (TGEV) results in an acute, severe pathology and high mortality in piglets, while attenuated TGEV only causes moderate clinical reactions. Dendritic cells (DCs), through uptake and presentation of antigens to T cells, initiate distinct immune responses to different infections. In this study, an attenuated TGEV (STC3) and a virulent TGEV (SHXB) were used to determine whether porcine DCs play an important role in pathogenetic differences between these two TGEVs. Our results showed that immature and mature monocyte-derived dendritic cells (Mo-DCs) were susceptible to infection with SHXB and STC3. However, only SHXB inhibited Mo-DCs to activate T-cell proliferation by down-regulating the expression of cell–surface markers and the secretion of cytokines in vitro. In addition, after 48h of SHXB infection, there was the impairment in the ability of porcine intestinal DCs to sample the antigen, to migrate from the villi to the lamina propria and to activate T-cell proliferation in vivo. In contrast, these abilities of intestinal DCs were enhanced in STC3-infected piglets. In conclusion, our results show that SHXB significantly impaired the functions of Mo-DCs and intestinal DCs in vitro and in vivo, while STC3 had the opposite effect. These differences may underlie the pathogenesis of virulent and attenuated TGEV in piglets, and could help us to develop a better strategy to prevent virulent TGEV infection.

Effects of porcine epidemic diarrhea virus on porcine monocyte-derived dendritic cells and intestinal dendritic cells.

Infection with porcine epidemic diarrhea virus (PEDV) causes damage to intestinal epithelial cells and results in acute diarrhea and dehydration with high mortality rates in swine. Dendritic cells (DCs) are highly effective antigen-presenting cells widely distributed beneath the intestinal epithelium, thus making them an early target for virus contact. DCs uptake and present viral antigens to T cells, which then initiate a distinct immune response. In this study, we investigated how attenuated PEDV (CV777) affects the function of porcine monocyte-derived dendritic cells (Mo-DCs). Our results show that the expression of Mo-DC surface markers such as SWC3a(+)CD1a(+), SWC3a(+)CD80/86(+) and SWC3a(+)SLA-II-DR(+) is increased after infection with CV777 for 24 h. Mo-DCs infected with CV777 produce higher levels of IL-12 and INF-γ compared to mock-infected Mo-DCs but the expression profile for IL-10 does not change. Interactions between Mo-DCs and CV777 significantly influence the stimulation of the T cell response in vitro. Consistent with these results, after 48 h of CV777 infection, there is enhancement in the ability of porcine intestinal DCs to sample the antigen and activate T-cell proliferation in vivo. The enhancement of sampling and presentation is most pronounced for immature Mo-DCs. These results suggest that CV777 stimulates the ability of Mo-DCs to sample and present antigen. We conclude that CV777 may be a useful vaccine to trigger adaptive immunity.

Bursopentin (BP5) protects dendritic cells from lipopolysaccharide-induced oxidative stress for immunosuppression.

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.

Retinoic acid facilitates inactivated transmissible gastroenteritis virus induction of CD8(+) T-cell migration to the porcine gut.

The digestive tract is the entry site for transmissible gastroenteritis virus (TGEV). TGEV transmission can be prevented if local immunity is established with increased lymphocytes. The current parenteral mode of vaccination stimulates systemic immunity well, but it does not induce sufficient mucosal immunity. Retinoic acid (RA) plays an important role in the induction of cells that imprint gut-homing molecules. We examined whether RA assist parenteral vaccination of pigs could improve mucosal immunity. We demonstrated that elevated numbers of gut-homing CD8+ T cells (which express α4β7 and CCR9 molecules) were presented in porcine inguinal lymph nodes and were recruited to the small intestine by RA. Intestinal mucosal immunity (IgA titre) and systemic immunity (serum IgG titre) were enhanced by RA. Therefore, we hypothesized that RA could induce DCs to form an immature mucosal phenotype and could recruit them to the small intestinal submucosa. Porcine T-cells expressed β7 integrin and CCR9 receptors and migrated to CCL25 by a mechanism that was dependent of activation by RA-pretreated DCs, rather than direct activation by RA. Together, our results provide powerful evidence that RA can assist whole inactivated TGEV (WI-TGEV) via subcutaneous (s.c.) immunization to generate intestinal immunity, and offer new vaccination strategies against TGEV.

Bacillus amyloliquefaciens SQR9 induces dendritic cell maturation and enhances the immune response against inactivated avian influenza virus.

The objective of this study was to evaluate the stimulatory effects of Bacillus amyloliquefaciens SQR9 on dendritic cells (DCs) and to verify its ability to enhance the immune response by modulating DC maturation. The results demonstrated that B. amyloliquefaciens SQR9 can adhere to the nasal epithelium and be taken up by DCs in the nasal mucosa, thereby inducing DC maturation and resulting in increased CD80, CD86, CD40 and MHCII expression and cytokine secretion. The frequencies of CD4+ and CD8+ T cells and CD69+ memory T cells were increased in spleens after nasal immunization with virus plus B. amyloliquefaciens SQR9 compared to immunization with inactivated H9N2 AIV alone. Moreover, the levels of sIgA in the nasal cavity, the trachea, and the lung and the levels of IgG, IgG1, and IgG2a in serum were significantly increased in mice administered WIV plus SQR9 compared to mice administered H9N2 WIV alone. The results of this study demonstrated that B. amyloliquefaciens SQR9 can stimulate DC maturation to effectively induce an immune response. In conclusion, an effective immune response may result from the uptake of H9N2 by DCs in the nasal mucosa, thereby stimulating DC maturation and migration to cervical lymph nodes to initiate immune response.