Invasion of bovine peripheral blood mononuclear cells and erythrocytes by Mycoplasma bovis

Abstract

Mycoplasma bovis is a small, cell wall-less bacterium that contributes to a number of chronic inflammatory diseases in both dairy and feedlot cattle, including mastitis and bronchopneumonia. Numerous reports have implicated M. bovis in the activation of the immune system, while at the same time inhibiting immune cell proliferation. However, it is unknown whether the specific immune-cell population M. bovis is capable of attaching to and potentially invading. Here, we demonstrate that incubation of M. bovis Mb1 with bovine peripheral blood mononuclear cells (PBMC) resulted in a significant reduction in their proliferative responses while still remaining viable and capable of gamma interferon secretion. Furthermore, we show that M. bovis Mb1 can be found intracellularly (suggesting a role for either phagocytosis or attachment/invasion) in a number of select bovine PBMC populations (T cells, B cells, monocytes, γδ T cells, dendritic cells, NK cells, cytotoxic T cells, and T-helper cells), as well as red blood cells, albeit it at a significantly lower proportion. M. bovis Mb1 appeared to display three main patterns of intracellular staining: diffuse staining, an association with the intracellular side of the cell membrane, and punctate/vacuole-like staining. The invasion of circulating immune cells and erythrocytes could play an important role in disease pathogenesis by aiding the transport of M. bovis from the lungs to other sites.

FIG. 1.

Invasion of bovine PBMC by M. bovis Mb1. Bovine PBMC from 12 beef cattle were incubated for various times (1, 2, 3, and 24 h) with M. bovis Mb1, and the percentage of M. bovis-infected PBMC was determined by a gentamicin resistance assay. (A) Viable M. bovis recovered. At 1, 2, and 3 h, M. bovis demonstrates a low-level invasion/infection of PBMC (between 5 and 10% of PBMC are infected). At 24 h, all PBMC are infected, and those with >100% infection are representative of more than 1 M. bovis organism per PBMC. The bars show the median of the values. After 24 h of incubation, the number of intracellular M. bovis was significantly higher (***, P < 0.001) than the 1-, 2-, and 3-h time points. (B) Viable bovine PBMC after incubation with M. bovis. C, control PBMC; I, PBMC infected with M. bovis. The bars show the median of the values. After 24 h of incubation with M. bovis, there was a significant decrease (***, P < 0.001) in PBMC viability.

FIG. 2.

M. bovis Mb1-induced IFN-γ secretion. Bovine PBMC were incubated with M. bovis Mb1 (MOIs of 0.1 and 1), and the number of IFN-γ-secreting cells was determined by an ELISPOT assay. M. bovis incubation with PBMC from four beef cattle resulted in a significant increase in the number of IFN-γ-secreting cells compared to the control. However, the response was not significantly enhanced by the combination of M. bovis and ConA. The bars show the median values. *, P < 0.05; **, P < 0.01.

FIG. 3.

M. bovis Mb1 inhibition of bovine PBMC proliferation. Compared to ConA, incubation of live, heat-killed, or culture supernatant of M. bovis Mb1 with bovine PBMC from 12 beef cattle with or without the addition of ConA resulted in the significant attenuation (***, P < 0.001) of PBMC proliferation. The dotted line shows the baseline level, while the bars show the median values.

FIG. 4.

M. bovis and apoptosis of bovine PBMC. Bovine PBMC were incubated with or without M. bovis Mb1 for either 24 h (A) or 72 h (B). Both early and late apoptotic events were monitored by flow cytometry. (A1) annexin V-FITC labeling of early apoptotic cells (81%) in bovine PBMC at 24 h under baseline conditions. (A2) Annexin V FITC and PI labeling of apoptotic cells displaying both late apoptotic and necrotic events (22%) in bovine PBMC at 24 h under baseline conditions. (A3) Annexin V-FITC labeling of early apoptotic cells (34%) in bovine PBMC incubated with M. bovis for 24 h. (A4) Annexin V-FITC and PI labeling of apoptotic cells displaying both late apoptotic and necrotic events (13%) in bovine PBMC incubated with M. bovis for 24 h. (B1) FlowTACS (DNA fragmentation) analysis of late apoptotic events (57%) in bovine PBMC at 72 h under baseline conditions. (B2) FlowTACS (DNA fragmentation) analysis of late apoptotic events (31%) in bovine PBMC incubated with M. bovis for 72 h.

FIG. 5.

Confocal z-scan of M. bovis Mb1 invasion of select bovine PBMC populations. All confocal images represent a scan (slice) through the cell, and therefore any staining within the cell membrane is considered internal. Bovine PBMC were incubated with R18-labeled M. bovis Mb1 (red color) for 2 h (A) and 24 h (B), respectively. Large red “structures” are indicative of a “clump” of M. bovis. Whether these specific structures are intracellular or external are unknown. The green color is indicative of select FITC-labeled PBMC populations. M. bovis was found intracellular in all cell types at 2 and 24 h, although the specific locale and degree of internalization varied both temporally and between the cell types themselves. In some cells, M. bovis appeared to primarily associate with the intracellular membrane (T cells; CD3, cytotoxic T cells; CD8, B cells; CD21). In others it displayed diffuse staining (T helper cells; CD4, monocytes; CD14), and in some cases it appeared to be contained within vacuolelike structures (cytotoxic T cells; CD8, NK cells; CD335). Each image is representative of four fields of view and three separate experiments.

FIG. 6.

Confocal z-scan of M. bovis Mb1-induced IFN-γ production in select PBMC populations. Bovine PBMC were incubated with R18-labeled M. bovis Mb1 (red color) for 24 h, and the select PBMC type-induced IFN-γ production was determined. The green color is indicative of select FITC-labeled PBMC populations, and the blue (Alexa 405) color is indicative of IFN-γ. The pink coloring observed in some cells indicates colocalization of M. bovis and IFN-γ. M. bovis induced IFN-γ production in CD3 (T cells), CD4 (T-helper cells), CD8 (cytotoxic T cells), CD335 (NK cells), and TcR1-N24 cells (γδ T cells).

FIG. 7.

Invasion of bovine RBCs by M. bovis Mb1. Bovine RBCs from 12 beef cattle were incubated for various times (1, 2, 3, and 24 h) with M. bovis Mb1 (MOI of 5), and the percentage of M. bovis-infected PBMC was determined by a gentamicin resistance assay. The bars show the median values. During the first 3 h, M. bovis was found to be primarily extracellular, with some M. bovis-infected PBMC occurring at the 3-h time point. However, after 24 h of incubation, ca. 20% of the PBMC were infected with M. bovis.