LncRNA H19/microRNA-675/PPARα axis regulates liver cell injury and energy metabolism remodelling induced by hepatitis B X protein via Akt/mTOR signalling Publication date: December 2019 Source: Molecular Immunology, Volume 116 Author(s): Yiqing Liu, Li Xu, Bingru Lu, Miaoqing Zhao, Li Li, Wenping Sun, Zhanjun Qiu, Bingchang Zhang Abstract Emerging evidence indicates that the lncRNAs/microRNA/mRNA axis plays important roles in a variety of diseases. This study was aimed to investigate the potential roles and underlying molecular mechanisms of lncRNA H19 and H19-derived miR-675 in regulating hepatitis B virus (HBV)-associated liver injury. mRNA and miR-675 levels were determined by quantitative real-time PCR (qRT-PCR), protein levels were determined by western blot, cell viability was measured by the MTT assay, cell apoptosis was measured by flow cytometry, inflammatory cytokine production was determined by ELISA, oxidative stress and energy metabolism were assessed by commercial kits, and the target relationship between PPARα and miR-675 was confirmed by the dual-luciferase reporter assay. The results showed that the expression of lncRNA H19 and miR-675 was up-regulated in patients with chronic hepatitis B (n = 20). Inhibition of lncRNA H19 or miR-675 in L02 cells increased cell viability, suppressed hepatitis B X protein (HBx)-induced cell apoptosis, inflammatory cytokine production, and oxidative stress, and remodelled energy metabolism. Furthermore, PPARα was found to be a target gene of miR-675. The expression of PPARα was down-regulated in patients with chronic hepatitis B, and there was a negative correlation between the expression of lncRNA H19 and PPARα, or between miR-675 and PPARα. Moreover, by knocking down the expression of PPARα, the actions (apoptosis, inflammatory factors, oxidative stress, and energy metabolism) of lncRNA H19 or miR-675 inhibition in HBx-induced L02 cells were at least partially reversed. In addition, HBx-induced elevated levels of p-AktSer473, p-AktThr308 and p-mTORSer2448 were down-regulated by lncRNA H19 or miR-675 inhibition. Furthermore, PPARα knockdown partly reversed the down-regulated effects of H19 or miR-675 inhibition. Taken together, these data indicate that the lncRNA H19/miR-675/PPARα axis regulates liver cell injury and energy metabolism remodelling induced by HBx, which may be related to the modulation of Akt/mTOR signalling.

Fugu, Takifugu ruberipes, mucus keratins act as defense molecules against fungi Publication date: December 2019 Source: Molecular Immunology, Volume 116 Author(s): Ko Shibuya, Shigeyuki Tsutsui, Osamu Nakamura Abstract Keratin is a cytoskeletal protein that constitutes the intermediate filament. Its distribution is restricted to epithelial tissues in mammals, but is wider in fish. An interesting feature of fish keratin is that it is abundant in the cutaneous mucus. However, the biological function of keratin in the mucus has not been explored. In the present study, we hypothesized that mucus keratins of fugu Takifugu rubripes function as antimicrobial molecules. To verify this hypothesis, we first identified all of the keratins expressed in the epidermis and present in mucus. Five of 15 keratins including Tr-K4 expressed in the epidermis were identified in the mucus. Subsequently, we examined the interaction of keratin molecules present in fugu mucus with yeast. Affinity chromatography using yeast as a carrier and subsequent LC–MS/MS analysis revealed that three types of keratin were bound to the yeast. Furthermore, yeast incubated with fugu mucus was agglutinated, and this was inhibited by anti-recombinant Tr-K4 (rTr-K4) antibody. Immunohistochemical analysis also revealed that keratin was attached to the surface of agglutinated yeasts. These findings indicate that mucus keratin agglutinates yeast. Furthermore, we found insoluble clumps in fugu mucus, which were mainly comprised of keratin. After incubation of yeast with soluble mucus fraction, insoluble clumps incorporating yeast were formed. This observation suggests that fugu mucus keratin sequesters microbes into insoluble clumps, which are eventually eliminated from the mucus. Here, we present our finding of the novel function of keratin as a defense molecule in fish mucus.

Critical roles of NLRP3 inflammasome in IL-1β secretion induced by Corynebacterium pseudotuberculosis in vitro Publication date: December 2019 Source: Molecular Immunology, Volume 116 Author(s): Zuoyong Zhou, Hexian Li, Shangquan Tian, Wenyi Yi, Yang Zhou, Haoyue Yang, Xiao Li, Bi Wu, Xiaoxia Li, Junjun Wu, Zhiying Wang, Shijun Hu, Rendong Fang Abstract Corynebacterium pseudotuberculosis is a prominent human and animal pathogen causing chronic inflammatory diseases. Interleukin-1β (IL-1β) is involved in the response to such pathogenic infections. However, the mechanism by which IL-1β is secreted during C. pseudotuberculosis infection remains unclear. This study aimed to investigate the mechanism underlying IL-1β secretion by macrophages infected with C. pseudotuberculosis. Herein, we firstly revealed that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Casp1) play critical roles in IL-1β secretion rather than IL-1β precursor (pro-IL-1β) expression in C. pseudotuberculosis-infected macrophages. Toll like receptor 4 (TLR4) is partially involved in IL-1β secretion, while absent in melanoma 2 (AIM2) is not involved in IL-1β secretion by C. pseudotuberculosis-infected macrophages. In addition, nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinases (p38 MAPK) inhibitors almost attenuated IL-1β secretion, implying that NF-κB and p38MAPK pathway are involved in IL-1β secretion in C. pseudotuberculosis-infected macrophages. Furthermore, C. pseudotuberculosis were significantly more numerous in Nlrp3−/−, Asc−/−, and Casp-1−/− macrophages than in WT macrophages at 24 h after infection (P < 0.05), indicating that NLRP3 inflammasome components limit C. pseudotuberculosis replication in macrophages. Together, these data provide novel insights into the mechanisms underlying IL-1β secretion in C. pseudotuberculosis-infected macrophages and further the current understanding of the host pro-inflammatory immune response against this pathogen.

EMCHD2019 - Abstracts Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s):

Corrigendum to “The Schistosoma mansoni cyclophilin a epitope 107–121 induces a protective immune response against schistosomiasis” [Mol. Immunol. 111(2019) 172–181] Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Tatiane Teixeira de Melo, Mariana Moreira Mendes, Clarice Carvalho Alves, Gardênia Braz Carvalho, Viviane Cristina Fernandes, Deborah Laranjeira Ferreira Pimenta, Marina de Moraes Mourão, Fatou Gai, Marina Kalli, Aline Coelho, Rosy Iara Maciel de Azambuja Ribeiro, Franco H. Falcone, Rosiane Aparecida da Silva Pereira, Cristina Toscano Fonseca

Antiviral activity of merimepodib against foot and mouth disease virus in vitro and in vivo Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Shi-fang Li, Mei-jiao Gong, Jun-jun Shao, Yue-feng Sun, Yong-guang Zhang, Hui-yun Chang Abstract Foot and mouth disease virus (FMDV), a member of family Picornaviridae, belongs to the genus Aphthovirus, which causes foot and mouth disease (FMD), a highly transmissible disease that affects cloven-hoof animals. In spite of the fact that efficient vaccines are available, effective antiviral molecules for FMD are needed to reduce viral infection during early stages of infection. In this study, merimepodib was found to efficiently inhibit FMDV replication in a dose-dependent manner. The 50% inhibitory concentration (IC50) of merimepodib antiviral activity against two distinct FMDV strains (O/MYA98/BY/2010 and A/GD/MM/CHA/2013) was estimated to be 7.859 and 2.876 μM, respectively, while the 50% cytotoxic concentration (CC50) of merimepodib was found to be 47.74 μM. Furthermore, treatment with 30 μg merimepodib efficiently prolonged the survival time of suckling mice infected with FMDV. Taken together, these results suggested that merimepodib has the potential to be a novel antiviral agent against FMDV.

Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Lisa Tuppo, Claudia Alessandri, Ivana Giangrieco, Michela Ciancamerla, Claudia Rafaiani, Maurizio Tamburrini, Maria Antonietta Ciardiello, Adriano Mari Abstract The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.

Immunoinformatics prediction of OMP2b and BCSP31 for designing multi-epitope vaccine against Brucella Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Zhiwei Li, Fengbo Zhang, Chuntao Zhang, Changmin Wang, Peipei Lu, Xiao Zhao, Lijun Hao, Jianbing Ding Abstract Brucella poses a serious threat to human health. High quality vaccines for Brucella are urgently needed to effectively reduce the incidence of brucellosis. OMP2b and BCSP31 are important component proteins of the Brucella outer membrane and are highly immunogenic. Here, we used the bioinformatics software ProtParam, SOPMA, SWISS-MODEL, Rasmol, BepiPred, SYFPEITHI and IEDB to analyze the structure of these two proteins and predict the epitopes of T cells and B cells. Through analysis, we predicted three Th cell epitopes, seven CTL epitopes, eight B cell epitopes, and one T-B combined epitope of OMP2b protein. Subsequently, we also obtained three Th cell epitopes, six CTL epitopes, nine B cell epitopes and one T-B combined epitope of BCSP31 protein. The T-B combined epitopes and CTL epitopes of OMP2b and those of BCSP31 were synthesized to detect their immunogenicity. The IFN-γ ELISPOT assay showed that the T-B combined epitope peptides of OMP2b and BCSP31 activated Th cell immune responses. ELISA analysis detected the specific antibodies against the T-B combined epitope peptide of OMP2b and BCSP31 in the serum of Brucellosis patients. Additionally, CTL epitope peptide of OMP2b and BCSP31 proteins promoted the secretion of soluble perforin and granzyme B in the culture supernatant. In conclusion, our study shows that the T-B combined epitopes and CTL epitopes of OMP2b and BCSP31 have immunogenicity and immunoreactivity. Our results may lay a theoretical foundation for the development of vaccines against Brucella.

B-cell epitopes: Discontinuity and conformational analysis Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Saba Ferdous, Sebastian Kelm, Terry S. Baker, Jiye Shi, Andrew C.R. Martin Abstract Peptide vaccines have many potential advantages over conventional ones including low cost, lack of need for cold-chain storage, safety and specificity. However, it is well known that approximately 90% of B-cell epitopes (BCEs) are discontinuous in nature making it difficult to mimic them for creating vaccines. In this study, the degree of discontinuity in B-cell epitopes and their conformational nature is examined. The discontinuity of B-cell epitopes is analyzed by defining ‘regions’ (consisting of at least three antibody-contacting residues each separated by ≤3 residues) and small fragments (antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm has been developed that classifies each region's shape as straight, curved or folded on the basis that straight and folded regions are more likely to retain their native conformation as isolated peptides. We have investigated the structures of 488 B-cell epitopes from which 1282 regions and 1018 fragments have been identified. 90% of epitopes have five or fewer regions and five or fewer fragments with 14% containing only one region and 4% being truly linear (i.e. having one region and no fragments). Of the 1282 regions, 508 are straight in shape, 626 are curved and 148 are folded.

Complement activation on neutrophils initiates endothelial adhesion and extravasation Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Antonina Akk, Luke E. Springer, Lihua Yang, Samantha Hamilton-Burdess, John D. Lambris, Huimin Yan, Ying Hu, Xiaobo Wu, Dennis E. Hourcade, Mark J. Miller, Christine T.N. Pham Abstract Neutrophils are essential to the pathogenesis of many inflammatory diseases. In the autoantibody-mediated K/BxN model of inflammatory arthritis, the alternative pathway (AP) of complement and Fc gamma receptors (FcγRs) are required for disease development while the classical pathway is dispensable. The reason for this differential requirement is unknown. We show that within minutes of K/BxN serum injection complement activation (CA) is detected on circulating neutrophils, as evidenced by cell surface C3 fragment deposition. CA requires the AP factor B and FcγRs but not C4, implying that engagement of FcγRs by autoantibody or immune complexes directly triggers AP C3 convertase assembly. The absence of C5 does not prevent CA on neutrophils but diminishes the upregulation of adhesion molecules. In vivo two-photon microscopy reveals that CA on neutrophils is critical for neutrophil extravasation and generation of C5a at the site of inflammation. C5a stimulates the release of neutrophil proteases, which contribute to the degradation of VE-cadherin, an adherens junction protein that regulates endothelial barrier integrity. C5a receptor antagonism blocks the extracellular release of neutrophil proteases, suppressing VE-cadherin degradation and neutrophil transendothelial migration in vivo. These results elucidate the AP-dependent intravascular neutrophil-endothelial interactions that initiate the inflammatory cascade in this disease model but may be generalizable to neutrophil extravasation in other inflammatory processes.