Browsing by Author "Zhao, Wenpeng"

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    Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro
    (2021-01-19) Shi, Yuxiang; Zhao, Wenpeng; Liu, Gang; Ali, Tariq; Chen, Peng; Liu, Yongxia; Kastelic, John P; Han, Bo; Gao, Jian
    Abstract Background Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro. Results Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1β concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h. Conclusions Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.
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    Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures
    (2021-12-11) Zhao, Wenpeng; He, Fumeng; Barkema, Herman W.; Xu, Siyu; Gao, Jian; Liu, Gang; Deng, Zhaoju; Shahid, Muhammad; Shi, Yuxiang; Kastelic, John P.; Han, Bo
    Abstract Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.