Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

Toxoplasma gondii eIF-5A Modulates the Immune Response of Murine Macrophages In Vitro.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can elicit a robust immune response during infection. Macrophage cells have been shown to play an important role in the immune response against T. gondii. In our previous study, the eukaryotic translation initiation factor 5A (eIF-5A) gene of T. gondii was found to influence the invasion and replication of tachyzoites. In this study, the recombinant protein of T. gondii eIF-5A (rTgeIF-5A) was incubated with murine macrophages, and the regulatory effect of TgeIF-5A on macrophages was characterized. Immunofluorescence assay showed that TgeIF-5A was able to bind to macrophages and partially be internalized. The Toll-like receptor 4 (TLR4) level and chemotaxis of macrophages stimulated with TgeIF-5A were reduced. However, the phagocytosis and apoptosis of macrophages were amplified by TgeIF-5A. Meanwhile, the cell viability experiment indicated that TgeIF-5A can promote the viability of macrophages, and in the secretion assays, TgeIF-5A can induce the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) from macrophages. These findings demonstrate that eIF-5A of T. gondii can modulate the immune response of murine macrophages in vitro, which may provide a reference for further research on developing T. gondii vaccines.

IFN-γ inhibitory molecules derived from Eimeria maxima inhibit IL-12 secretion by modulating MAPK pathways in chicken macrophages.

IFN-γ plays a crucial role in resisting intracellular parasitic protozoa, such as Eimeria species. In our previous study, we identified 4 molecules derived from Eimeria maxima (E. maxima) that significantly inhibited IFN-γ production. However, the mechanism underlying this inhibitory effect remains unknown. In this study, we first investigated the effects of these 4 IFN-γ inhibitory molecules on the expression levels of chicken Toll-like receptors (chTLRs), IL-12, IL-10, TGF-ß, and TNF-α in chicken macrophage HD11 and bone marrow-derived dendritic cells (BMDCs). The results demonstrated that these 4 inhibitory molecules significantly downregulated the mRNA levels of chTLR-2, chTLR-4, chTLR-21, and both mRNA and protein levels of IL-12. Subsequently, to clarify the effects of these 4 inhibitory molecules on the IL-12 secretion-related signaling pathways in chicken macrophages, qRT-PCR and Western blot were used to detect the changes of key molecules involved in the signaling pathways of IL-12 secretion (NF-κB, ERK1/2, p38, JNK, STAT3) following coincubation with these inhibitory molecules. Finally, RNAi was employed to verify the function of key molecules in the signaling pathway. The results revealed a significant upregulation in the expression of ERK1/2 phosphorylated protein induced by the 4 inhibitory molecules. Knockdown of the ERK1/2 gene significantly reduced the inhibitory effect of the 4 E. maxima inhibitory molecules on IL-12. These findings indicate that the 4 inhibitory molecules can inhibit the secretion of IL-12 by upregulating the expression of ERK1/2 phosphorylated protein, which is a key molecule in the ERK-MAPK pathway. Our study may contribute to elucidating the mechanisms underlying immune evasion during E. maxima infections, thereby providing new insights for the control of chicken coccidiosis.

Molecular Docking and In Silico Simulation of Trichinella spiralis Membrane-Associated Progesterone Receptor Component 2 (Ts-MAPRC2) and Its Interaction with Human PGRMC1.

Background. Trichinellosis is a foodborne zoonotic disease caused by Trichinella spp., including Trichinella spiralis. This parasitic disease ranks as seven of the most infectious in the world. In this context, it is important to develop a vaccine that can combat Trichinellosis, especially for humans and pigs. This would be an important step in preventing transmission. In this study, we focus on homology modelling, binding site prediction, molecular modelling, and simulation techniques used to explore the association between Trichinella spiralis membrane-associated progesterone receptor component 2 (Ts-MAPRC2) and the human PGRMC1 protein. It was found that the progesterone receptor component 2 of T. spiralis has 44.54% sequence identity with human PGRMC1 (PDB ID: 4X8Y). Binding sites predicted for human PGRMC1 are GLU 7, PHE 8, PHE 10, PHE 18, LEU 27, ASP 36, and VAL 104. Molecular docking has six clusters based on Z scores. They range from -1.5 to 1.8. It was found that the progesterone receptor component 2 of T. spiralis has 44.54% sequence identity with human PGRMC1. During simulation, the average RMSD was 2.44 ± 0.20 Å, which indicated the overall stability of the protein. Based on docking studies and computational simulations, we hypothesized that the interaction of the proteins Trichinella spiralis membrane-associated progesterone receptor component 2 and human PGRMC1 formed stable complexes. The discovery of Ts-MAPRC2 may pave the way for the development of drugs and vaccines to treat Trichinellosis.

Immunological characterization of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α.

Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of the TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long-missed avian ortholog, and its immunological properties remain largely unknown compared to those of its mammalian counterparts. Here, we report the functional characterization and immunomodulatory properties of chTNF-α using a panel of newly developed anti-chTNF-α mouse monoclonal antibodies (mAbs). Using anti-chTNF-α mAbs, we determined the tissue expression of chTNF-α in lymphoid and non-lymphoid organs. A chTNF-α-specific antigen-capture sandwich ELISA was developed using compatible mAb partners by screening and validation of ten different mAbs. Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens, Eimeria, or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expression of chTNF-α in primary immune cells or cell lines derived from chickens was validated by immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Notably, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results enhance our understanding of the functional characteristics of chTNF-α, and these anti-chTNF-α mAbs will serve as valuable immune reagents to inform on inflammatory responses and disease pathogenesis in the fundamental and applied studies of avian species.

Immunological studies on chicken interferon-kappa using an antigen-capture ELISA developed using new mouse monoclonal antibodies.

Interferon (IFN)-κ is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. In this study, we developed an antigen-capture ELISA using newly developed mouse monoclonal antibodies (mAbs) which are specific for chicken IFN-κ (chIFN-κ) and showed that this ELISA can measure native chIFN-κ production during the activation of macrophages by polyinosinic:polycytidylic acid (poly I:C). The recombinant chicken IFN-κ expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-κ were selected and characterized based on their specificity and binding activity toward chIFN-κ via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-κ, two sets of the best capture and detection mAbs combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-κ was induced in chicken HD11 macrophages using poly I:C. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-κ in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-κ mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 fibroblast cells stimulated with recombinant chIFN-κ proteins. All five mAbs blocked the mRNA expression of ISGs in a dose-dependent manner. Our results validate the specificity and utility of these newly developed mAbs for the detection of native chicken IFN-κ. This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-κ in the normal and diseased states.

Proteomics analysis reveals that the proto-oncogene eIF-5A indirectly influences the growth, invasion and replication of Toxoplasma gondii tachyzoite.

BACKGROUND: The proliferative stage (tachyzoite) of Toxoplasma gondii (T. gondii) is critical for its transmission and pathogenesis, and a proto-oncogene eukaryotic translation initiation factor (eIF-5A) plays an important role in various cellular processes such as cell multiplication. METHODS: We performed a proteomic study to evaluate the specific roles of eIF-5A involved in invasion and replication of T. gondii, and both in vivo and in vitro trials using eIF-5A-interfered and wild tachyzoites were performed to verify the proteomic results. RESULTS: The results of our study showed that T. gondii eIF-5A affected tachyzoite growth and also participated in the synthesis of proteins through regulation of both ribosomal and splicing pathways. Inhibition of eIF-5A in T. gondii resulted in the downregulated expression of soluble adhesions, such as microneme protein 1 (MIC1) and MIC4, which in turn decreased the parasite population that adhered to the surface of host cells. The reduced attachment, combined with lower expression of some rhoptry proteins (ROPs) and dense granule antigens (GRAs) involved in different stages of T. gondii invasion such as ROP4 and GRA3, ultimately reduce the invasion efficiency. These processes regulated by eIF-5A eventually affect the replication of tachyzoites. CONCLUSIONS: Our findings showed that eIF-5A influenced tachyzoite survival and was also involved in the process of parasite invasion and replication. These results will provide new clues for further development of targeted drugs to control T. gondii infection.

Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4.

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro.

BACKGROUND: Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood. METHODS: Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry. RESULTS: The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose-dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered. CONCLUSIONS: Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.

Toxoplasma gondii Histone 4 Affects Some Functions of Murine Ana-1 Macrophages In Vitro.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can infect almost all nucleated cells. Histone proteins and DNA form the nucleosomes, which are the fundamental building blocks of eukaryotic chromatin. Histone 4 is an essential component of a histone octamer. In the present study, T. gondii histone 4 (TgH4) was cloned and the regulatory effect of TgH4 on murine macrophages was characterized. Bioinformatics analysis revealed that TgH4 was highly conserved in structure. Recombinant TgH4 (rTgH4) protein was identified by sera from rats experimentally infected with T. gondii and native TgH4 in the total soluble protein of T. gondii tachyzoites was recognized by polyclonal antibodies against rTgH4, as indicated by immunoblotting analysis. Immunofluorescence assay showed that TgH4 binds to macrophages. Following incubation with rTgH4, the toll-like receptor 4 (TLR4) level of the macrophages was downregulated. Meanwhile, chemotaxis and the proliferation of macrophages were inhibited. However, rTgH4 can promote phagocytosis, apoptosis, and the secretion of nitric oxide, interleukin-6, and tumor necrosis factor-α from macrophages. Just 80 µg/ml rTgH4 can significantly elevate the secretion of interleukin-10 and interleukin-1ß (p < 0.05 and p < 0.01). Viewed together, these outcomes indicated that rTgH4 can affect the functions of murine macrophages in vitro.

Effects of Recombinant Toxoplasma gondii Citrate Synthase I on the Cellular Functions of Murine Macrophages In vitro.

Toxoplasmosis, which is one of the most widespread zoonoses worldwide, has a high incidence and infection can result in severe disease in humans and livestock. Citrate synthase (CS) is a component of nearly all living cells that plays a vital role in the citric acid cycle, which is the central metabolic pathway of aerobic organisms. In the present study, the citrate synthase I gene of Toxoplasma gondii (T. gondii) (TgCSI) was cloned and characterized. The TgCSI gene had an open reading frame of 1665 bp nucleotides encoding a 555 amino acid protein with a molecular weight of 60 kDa. Using western blotting assay, the recombinant protein was successfully recognized by the sera of rats experimentally infected with T. gondii, while the native protein in the T. gondii tachyzoites was detected in sera from rats immunized with the recombinant protein of TgCSI. Binding of the protein to murine macrophages was confirmed by immuno fluorescence assay. Following incubation of macrophages with rTgCSI, the rTgCSI protein was found to have a dual function, with low concentrations (5-10 µg/mL) enhancing phagocytosis and high levels (80 µg/mL) inhibiting phagocytosis. Investigation of murine macrophage apoptosis illustrated that 5 µg/mL rTgCSI protein can significantly induce early apoptosis and late stage apoptosis (*p < 0.05), while 10 µg/mL rTgCSI protein significantly induced early apoptosis, but had no effect on late stage of apoptosis (**p < 0.01), and 80 µg/mL rTgCSI protein inhibited late stage apoptosis of macrophages (*p < 0.05). Cytokine detection revealed that the secretion of interleukin-10, interleukin-1ß, transforming growth factor-ß1 and tumor necrosis factor-α of macrophages increased after the cells were incubated with all concentration of rTgCSI, with the exception that 5 µg/mL rTgCSI had no effect on the secretion of interleukin-10 and interleukin-1ß. However, secretion of NO and cell proliferation of the macrophages were substantially reduced. Taken together, these results suggested that TgCSI can affect the immune functions of murine macrophages by binding to the cells in vitro.

Paeonol suppresses solar ultraviolet-induced skin inflammation by targeting T-LAK cell-originated protein kinase.

Excessive exposure to solar UV (SUV) is related with numerous human skin disorders, such as skin inflammation, photoaging and carcinogenesis. T-LAK cell- originated protein kinase (TOPK), an upstream of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinases (JNKs), plays an important role in SUV -induced skin inflammation, and targeting TOPK has already been a strategy to prevent skin inflammation. In this study, we found that the expression of TOPK, phosphorylation of p38 or JNKs was increased in human solar dermatitis tissues. The level of phosphorylation of p38 or JNKs increased in a dose and time dependent manner in HaCat cells or JB6 Cl41 cells after SUV treatment. Paeonol is an active component isolated from traditional Chinese herbal medicines, and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay showed that it has no toxicity to cells. Microscale thermophoresis (MST) assay showed that paeonol can bind TOPK ex vivo. In vitro kinase assay showed paeonol can inhibit TOPK activity. Ex vivo studies further showed paeonol suppressed SUV-induced phosphorylation level of p38, JNKs, MSK1 and histone H2AX by inhibiting TOPK activity in a time and dose dependent manner. Paeonol inhibited the secretion of IL-6 and TNF-α in HaCat and JB6 cells ex vivo. In vivo studies demonstrated that paeonol inhibited SUV-induced increase of TOPK, the phosphorylation of p38, JNKs and H2AX, and the secretion of IL-6 and TNF-α in Babl/c mouse. In summary, our data indicated a protective role of paeonol against SUV-induced inflammation by targeting TOPK, and paeonol could be a promising agent for the treatment of SUV-induced skin inflammation.

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips.

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.

Salidroside suppresses solar ultraviolet-induced skin inflammation by targeting cyclooxygenase-2.

Solar ultraviolet (SUV) irradiation causes skin disorders such as inflammation, photoaging, and carcinogenesis. Cyclooxygenase-2 (COX-2) plays a key role in SUV-induced skin inflammation, and targeting COX-2 may be a strategy to prevent skin disorders. In this study, we found that the expression of COX-2, phosphorylation of p38 or JNKs were increased in human solar dermatitis tissues and SUV-irradiated human skin keratinocyte HaCaT cells and mouse epidermal JB6 Cl41 cells. Knocking down COX-2 inhibited the production of prostaglandin E2 (PGE2), the phosphorylation of p38 or JNKs in SUV-irradiated cells, which indicated that COX-2 is not only the key enzyme for PGs synthesis, but also an upstream regulator of p38 or JNKs after SUV irradiation. The virtual ligand screening assay was used to search for natural drugs in the Chinese Medicine Database, and indicated that salidroside might be a COX-2 inhibitor. Molecule modeling indicated that salidroside can directly bind with COX-2, which was proved by in vitro pull-down binding assay. Ex vivo studies showed that salidroside has no toxicity to cells, and inhibits the production of PGE2, phosphorylation of p38 or JNKs, and secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) caused by SUV irradiation. In vivo studies demonstrated that salidroside attenuates the skin inflammation induced by SUV. In brief, our data provided the evidences for the protective role of salidroside against SUV-induced inflammation by targeting COX-2, and salidroside might be a promising drug for the treatment of SUV-induced skin inflammation.

Proteomic analysis of Eimeria acervulina sporozoite proteins interaction with duodenal epithelial cells by shotgun LC-MS/MS.

Although it has been known for many years that Eimeria acervulina (E. acervulina) initiates infection by invading the duodenal epithelial cells of chicken, the key protein molecules and the mechanisms of the parasite in invading are unknow. In this study, we found that 85 proteins of E. acervulina could bind with the chicken duodenal epithelial cells from Eimeria protein database. Among them, sixteen were identified only in Eimeria spp. correlation with invasion and evasion and 69 proteins were found in Eimeria spp. with more than 2 unique pep count. Nine out of the 16 proteins and 41 out of the 69 proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Most of the 9 annotated proteins occurred in binding, catalytic activity and cellular process whereas, 29 (70.73%) out of the 41 proteins had binding activity and 20 proteins (48.78%) had catalytic activity. The findings provided an insight into the interactive relationship between E. acervulina and host cells and will shed new lights on the understanding of molecular mechanisms of E. acervulina invasion and pathogenesis.