IntroductionBovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) pathway in immunosuppression in bovine mycoplasmosis.MethodsIn the initial experiments, we used enzyme‐linked immunosorbent assay to measure interferon‐γ (IFN‐γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis.ResultsExpectedly, IFN‐γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD‐1+CD4+ and PD‐L1+CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD‐1+CD4+ and PD‐1+CD8+ T cells were negatively correlated with IFN‐γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD‐1/PD‐L1 pathway in vitro by anti‐bovine PD‐1‐ and anti‐bovine PD‐L1 antibodies significantly upregulated the production of IFN‐γ from anti‐mycoplasma‐specific cells.ConclusionsThese results suggest that the PD‐1/PD‐L1 pathway could be involved in immune exhaustion of bovine mycoplasma‐specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E 2 (PGE 2 ) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE 2 in immune dysfunction and the relationship between PGE 2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE 2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE 2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE 2 levels were positively correlated with the proportions of PD-L1 + monocytes in M. bovis-infected cattle. Additionally, plasma PGE 2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE 2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade Goto et al.PGE 2 -Induced Immunodysfunction During Bovine Mycoplasmosis assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE 2 and the PD-1/PD-L1 pathway.

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Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1β, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.