Huahua Du

Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims:  To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles.

Iron Reduces M1 Macrophage Polarization in RAW264.7 Macrophages Associated with Inhibition of STAT1.

Iron metabolism in inflammation has been mostly characterized in macrophages exposed to pathogens or inflammatory conditions. The aim of this study is to investigate the cross-regulatory interactions between M1 macrophage polarization and iron metabolism. Firstly, we characterized the transcription of genes related to iron homeostasis in M1 RAW264.7 macrophages stimulated by IFN-γ. The molecular signature of M1 macrophages showed high levels of iron storage (ferritin), a low level of iron export (ferroportin), and changes of iron regulators (hepcidin and transferrin receptors), which favour iron sequestration in the reticuloendothelial system and are benefit for inflammatory disorders. Then, we evaluated the effect of iron on M1 macrophage polarization. Iron significantly reduced mRNA levels of IL-6, IL-1β, TNF-α, and iNOS produced by IFN-γ-polarized M1 macrophages. Immunofluorescence analysis showed that iron also reduced iNOS production. However, iron did not compromise but enhanced the ability of M1-polarized macrophages to phagocytose FITC-dextran. Moreover, we demonstrated that STAT1 inhibition was required for reduction of iNOS and M1-related cytokines production by the present of iron. Together, these findings indicated that iron decreased polarization of M1 macrophages and inhibited the production of the proinflammatory cytokines. The results expanded our knowledge about the role of iron in macrophage polarization.

Evidence for cell apoptosis suppressing white spot syndrome virus replication in Procambarus clarkii at high temperature

In shrimp, higher water temperatures (~32°C) can suppress the ability of white spot syndrome virus (WSSV) to replicate and cause mortality, but the mechanisms remain unclear. To investigate whether cell apoptosis might be involved, a Tdt-mediated dUTP nick-end label (TUNEL) method was used to assess levels of chromosomal DNA fragmentation in hepatopancreas and gill cells of Procambarus clarkii crayfish infected with WSSV and maintained at either 32 ± 1°C or 24 ± 1°C. Based on relative cell numbers with yellow-green colored TUNEL-positive nuclei, the apoptotic index was elevated in WSSV-infected crayfish maintained at 32°C. In gill tissue sections examined by transmission electron microscope, cells with nuclei displaying apoptotic bodies or marginated, condensed and fragmented chromatin without concurrent cell cytoplasm damage were also more prevalent. Flow cytometry sorting of annexin-stained cells showed apoptosis to be most prevalent in granular haemocytes, and assays for caspase-3 activity showed it to be most elevated in hepatopancreas tissue. Despite these indicators of cell apoptosis but consistent with WSSV replication being restricted at elevated temperatures, no increases in transcription of the viral anti-apoptosis genes ORF390 and ORF222 were detected by RT-PCR in shrimp maintained at 32°C, possibly due to the elevated levels of cellular apoptosis.

Synthetic Porcine Hepcidin Exhibits Different Roles in Escherichia coli and Salmonella Infections

ABSTRACT Hepcidin, an antimicrobial peptide, was discovered to integrate diverse signals from iron status and an infection threat and orchestrate a series of host-protective responses. Several studies have investigated the antimicrobial role of hepcidin, but the results have been controversial. Here, we aimed to examine the role of hepcidin in bacterial adherence and invasion in vitro. We found that porcine hepcidin could decrease the amount of the extracellular pathogen enterotoxigenic Escherichia coli (ETEC) K88 that adhered to cells because it caused the aggregation of the bacteria. However, addition of hepcidin to macrophages infected with the intracellular pathogen Salmonella enterica serovar Typhimurium enhanced the intracellular growth of the pathogen through the degradation of ferroportin, an iron export protein, and then the sequestration of intracellular iron. Intracellular iron was unavailable by use of the iron chelator deferiprone (DFO), which reduced intracellular bacterial growth. These results demonstrate that hepcidin exhibits different functions in extracellular and intracellular bacterial infections, which suggests that different defense strategies should be taken to prevent bacterial infection.

Iron Promotes Intestinal Development in Neonatal Piglets

Early nutrition is key to promoting gut growth and education of the immune system. Although iron deficiency anemia has long been recognized as a serious iron disorder, the effects of iron supplementation on gut development are less clear. Therefore, using suckling piglets as the model for iron deficiency, we assessed the impacts of iron supplementation on hematological status, gut development, and immunity improvement. Piglets were parenterally supplied with iron dextran (FeDex, 60 mg Fe/kg) by intramuscular administration on the third day after birth and slaughtered at the age of two days, five days, 10 days, and 20 days. It was expected that iron supplementation with FeDex improved the iron status with higher levels of serum iron, ferritin, transferrin, and iron loading in the liver by regulating the interaction of hepcidin and ferroportin (FPN). FeDex supplementation increased villus length and crypt depth, attenuated the pathological status of the duodenum, and was beneficial to intestinal mucosa. FeDex also influenced the intestinal immune development by stimulating the cytokines’ production of the intestine and enhancing the phagocytotic capacity of monocytes. Overall, the present study suggested that iron supplementation helped promote the development of the intestine by improving its morphology, which maintains its mucosal integrity and enhances the expression of immuno-associated factors.

Lipocalin 2 regulates intestine bacterial survival by interplaying with siderophore in a weaned piglet model of Escherichia coli infection

Iron is an essential nutrient that facilitates cell proliferation and growth, which plays a pivotal role in modulating the battle for survival between mammalian hosts and their pathogens. Pathogenic bacteria secrete siderophores to acquire iron from the host. However, lipocalin 2 (Lcn2), a siderophore-binding antimicrobial protein, binds to siderophores to prevent bacterial uptake of iron, which is critical for the control of systemic infection with Escherichia coli (E. coli). But few studies focus on the anti-infective response of Lcn2 in the intestines by inhibiting bacterial proliferation based on microbial iron metabolism. In this study, we showed that iron was sequestrated within cells in a piglet model of E. coli K88 infection. Siderophores was produced following E. coli K88 infection and siderophore-related genes expression was upregulated in iron-deficiency environment in vitro. Meanwhile, we found that Lcn2 expression was rapidly and robustly induced in jejunum by E. coli K88 infection and could be stimulated by IL-17 and IL-22. Furthermore, both Lcn2 induced in epithelial cells IPEC-1 and added exogenously as a recombinant protein could inhibit the growth of E. coli. We can conclude that Lcn2 is a crucial component of mucosal immune defense against intestinal infection with E. coli K88.Iron is an essential nutrient that facilitates cell proliferation and growth, which plays a pivotal role in modulating the battle for survival between mammalian hosts and their pathogens. Pathogenic bacteria secrete siderophores to acquire iron from the host. However, lipocalin 2 (Lcn2), a siderophore-binding antimicrobial protein, binds to siderophores to prevent bacterial uptake of iron, which is critical for the control of systemic infection with Escherichia coli (E. coli). But few studies focus on the anti-infective response of Lcn2 in the intestines by inhibiting bacterial proliferation based on microbial iron metabolism. In this study, we showed that iron was sequestrated within cells in a piglet model of E. coli K88 infection. Siderophores was produced following E. coli K88 infection and siderophore-related genes expression was upregulated in iron-deficiency environment in vitro. Meanwhile, we found that Lcn2 expression was rapidly and robustly induced in jejunum by E. coli K88 infection and could be stimulated by IL-17 and IL-22. Furthermore, both Lcn2 induced in epithelial cells IPEC-1 and added exogenously as a recombinant protein could inhibit the growth of E. coli. We can conclude that Lcn2 is a crucial component of mucosal immune defense against intestinal infection with E. coli K88.