Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis

Abstract

Introduction: Bovine 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.

Methods: In 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.

Results: Expectedly, 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.

Conclusions: These 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.

Keywords: Bovine mycoplasmosis; IFN-γ; PD-1; PD-L1; immunosuppression.

Figure 1

IFN‐γ production in bovine mycoplasmosis. IFN‐γ production was determined in supernatant of the PBMCs culture from mycoplasmosis‐afflicted and healthy control Holstein cattle by ELISA assay. PBMCs were incubated with the combination of anti‐bovine CD3 and anti‐bovine CD28 antibodies. Each line indicates the median production in each group.

Figure 2

Expression analysis of PD‐1 and PD‐L1 in bovine mycoplasmosis. Flow cytometric analysis of the PD‐1 expression on CD4⁺ T cells (A), CD8⁺ T cells (B), and PD‐L1 expression on CD11b⁺CD14⁺ monocytes (C) in PBMCs from cattle with mycoplasmosis and healthy control cattle. Each line indicates the median percentage in each group.

Figure 3

Comparative analysis of PD‐1 and PD‐L1 expression among bovine mycoplasmosis cases with different symptoms. PBMCs from M. bovis‐infected cattle with otitis media (n = 7), pneumonia (n = 2), and arthritis (n = 8) were analyzed.

Figure 4

Negative correlation between the proportion of PD‐1⁺ T cells and IFN‐γ production. IFN‐γ production in cattle with mycoplasmosis corresponds to that in Figure 1A (n = 15). Correlation statistics were analyzed using Spearman's correlation.

Figure 5

Enhancement of IFN‐γ production by anti‐PD‐1 and anti‐PD‐L1 mAbs in PBMCs from cattle with mycoplasmosis. PBMCs were cultivated with rat IgG control, anti‐PD‐1 mAb (A: 20 µg/mL), or anti‐PD‐L1 mAb (B: 20 µg/mL) in the presence of heat‐killed M. bovis. IFN‐γ production was measured by ELISA assay. Statistical comparisons between rat IgG control and blocking mAb were performed using Wilcoxon's matched‐pairs test.