Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-\u03baB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures

Wenpeng Zhao,Jian Gao,Bo Han,Gang Liu,Siyu Xu,Zhaoju Deng,Herman W Barkema,John P Kastelic,Yuxiang Shi,Fumeng He,Muhammad Shahid

doi:10.1186/s13567-021-01014-9

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.

Highlights

Mastitis is common in dairy cattle worldwide, causing serious reductions in milk yield and quality and large financial losses [1, 2]
P. bovis and P. ciferrii decreased bovine mammary epithelial cells (bMECs) viability at 12 h post infection, P. bovis caused a more profound decrease than P. ciferrii in the viability of bMECs (Figure 3D)
Infection of bMECs with Prototheca spp., especially P. bovis, damaged mitochondria and promoted Mitochondrial reactive oxygen species (mtROS) accumulation, which activated an inflammatory response through the NF-κB and NLRP3 inflammasome pathways and enhanced IL-1β production

Summary

Introduction

Mastitis is common in dairy cattle worldwide, causing serious reductions in milk yield and quality and large financial losses [1, 2]. The inflammasome is an upstream regulatory mechanism that triggers an inflammatory response when stimulated by pathogens. NLRP3 signaling usually confers protection, excessive activation can damage cells and cause inflammatory diseases [16,17,18]. The NLRP3 inflammasome can be generated and activated by Escherichia coli and Staphylococcus aureus, causing an aggravated inflammatory response and damage in bMECs [19, 20]. NF-κB participates, and has an important regulatory role, in activation of the NLRP3 inflammasome, which triggers an inflammatory response [21]. An activated NF-κB pathway could function as an upstream activator of NLRP3 and contribute to regulating inflammatory cytokines [22, 23]

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Results

Conclusion