Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

Abstract

Background: Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.

Results: Intracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFalpha at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFalpha compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565.

Conclusion: Taken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease.

Figure 1

IL-10 mRNA expressed by MDM cells exposed to M. paratuberculosis strains over time was measured by Real time RT PCR and fold change in gene expression relative to β actin was calculated by 2^-ΔΔCT method. Mean fold change in gene expression is plotted on Y-axis (note that the Y1-axis scales have been optimized for magnitude of IL-10 expression for each strain). Intracellular bacterial numbers based on the amplification of hsp65 were calculated based on genome size of MAP and represented as genome equivalents (GE) on second Y-axis. MDMs stimulated with B1018 and Hu6 MAP isolates increased IL-10 mRNA by 96 hrs PI relative to cell stimulations by S7565 MAP isolate and M. avium.

Figure 2

IL-10 protein secreted by MDM cells exposed to MAP over time was measured by ELISA. Total amount of protein (pg/ml) is plotted on Y-axis (note that the Y1-axis scales have been optimized for magnitude of IL-10 expression for each strain). Intracellular bacterial numbers were calculated based on genome size of MAP and represented as genome equivalents (GE) on second Y-axis. MDMs stimulated with B1018 and Hu6 MAP isolates gradually up regulated IL-10 secretion from 2-hr until 96-hr PI.

Figure 3

TNFα mRNA expressed by MDM cells exposed to MAP over time was measured by Real time RT PCR and fold change in gene expression relative to β actin was calculated by 2^-ΔΔCT method. Mean fold change in gene expression is plotted on Y-axis (note that the Y1-axis scales have been optimized for magnitude of IL-10 expression for each strain). Intracellular bacterial numbers based on the amplification of hsp65 were calculated based on the genome size of MAP and represented as genome equivalents (GE) on second Y-axis. MDMs stimulated with B1018 expressed lower levels of TNFα mRNA relative to other cell stimulations.

Figure 4

TNFα protein secreted by MDM cells exposed to MAP over time was measured by ELISA. Total amount of protein (pg/ml) is plotted on Y-axis (note that the Y1-axis scales have been optimized for magnitude of TNFα expression for each strain). Intracellular bacterial numbers were calculated based on the genome size of MAP and represented as genome equivalents (GE) on second Y-axis. MDMs stimulated with B1018 secreted low amounts of TNFα protein relative to other cell stimulations.