Mycoplasma hyopneumoniae Infection Activates the NOD1 Signaling Pathway to Modulate Inflammation

Abstract

Mycoplasma hyopneumoniae is a highly contagious pathogen causing porcine enzootic pneumonia, which elicits prolonged inflammatory response modulated by pattern recognition receptors (PRRs). Although significant advances have been achieved in understanding the Toll-Like receptors that recognize M. hyopneumoniae, the role of nucleotide-binding oligomerization domain 1 (NOD1) in M. hyopneumoniae infected cells remains poorly understood. This study revealed that M. hyopneumoniae activates the NOD1-RIP2 pathway and is co-localized with host NOD1 during infection. siRNA knockdown of NOD1 significantly impaired the TRIF and MYD88 pathway and blocked the activation of TNF-α. In contrast, NOD1 overexpression significantly suppressed M. hyopneumoniae proliferation. Furthermore, we for the first time investigated the interaction between M. hyopneumoniae mhp390 and NOD1 receptor, and the results suggested that mhp390 and NOD1 are possibly involved in the recognition of M. hyopneumoniae. These findings may improve our understanding of the interaction between PRRs and M. hyopneumoniae and the function of NOD1 in host defense against M. hyopneumoniae infection.

Keywords: Mycoplasma hyopneumoniae; NOD1; inflammation; interaction.; mhp390.

Figure 1

M. hyopneumoniae infection triggers NOD1 transcription and steadily up-regulates NOD1 protein abundance. (A) M. hyopneumoniae infection up-regulates NOD1 protein abundance. (B) M. hyopneumoniae infection triggers NOD1 transcription. ***P < 0.001 indicate statistically significant differences among different groups.

Figure 2

Knockdown of NOD1 inhibits the activation of RIP2 signaling pathway by M. hyopneumoniae. (A) Silencing efficiency of siRNA targeting NOD1. Alveolar macrophages were transfected with the indicated siRNA. The expression of NOD1 was evaluated by western blot at 24 h post transfection. Anti-β-actin was included as a control for sample loading. The relative levels of NOD1 in comparison to those in NC siRNA treated cells are shown as folds in the right panel. (B) The mRNA transcription of NOD1 was measured through real time RT-PCR and compared with that of GAPDH at 12 h/24 h post infection (hpi). (C) Alveolar macrophages were transfected with NOD1 siRNA or NC siRNA for 24 h, and then infected with M. hyopneumoniae (100 μL, 10⁸ CCU). Cells were collected at 6 hpi, and subjected to real-time RT-PCR to determine the expression of RIP2 mRNA. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences among different groups, and ns represents no difference.

Figure 3

M. hyopneumoniae infection activates NOD1-mediated TRIF and MYD88 signaling pathways. M. hyopneumoniae infection induces TRIF (A) and MYD88 (B) transcription in a time-dependent manner. Alveolar macrophages were infected with M. hyopneumoniae (100 μL, 10⁸ CCU) or mock-infected. Cells were collected separately at the indicated time points, and subjected to qRT-PCR to analyze the relative mRNA transcription. Alveolar macrophages were transfected with NOD1 siRNA or NC siRNA for 24 h, and the cells were then mock infected or infected with M. hyopneumoniae (100 μL, 10⁸ CCU). The cells were collected at 12 hpi. The MYD88 and TRIF mRNA expression levels were determined by qRT-PCR assay. (C) Knockdown of NOD1 blocked M. hyopneumoniae-induced mRNA expression of TNF-α. Transfection and infection experiments performed as described for panels (A, B) The cells were collected at 12 hpi and subjected to qRT-PCR to determine the mRNA expression. *P < 0.05, and ***P < 0.001 indicate statistically significant differences among different groups, and ns represents no difference.

Figure 4

NOD1 inhibits M. hyopneumoniae replication during infection. (A) Alveolar macrophages were transfected with pCMV-HA-NOD1 plasmid or empty pCMV-HA vector. At 0, 6, 12, 24, or 36 hpt, the cells were lysed and subjected to western blotting analysis. Relative expression levels of NOD1 are presented as folds in the right panel. (B) Cells were transfected with pCMV-HA-NOD1 or pCMV-HA for 24 h, and then infected with M. hyopneumoniae (100 μL, 10⁸ CCU). (C) Cells were transfected with Nc siRNA or si2815 for 24 h, and then infected with M. hyopneumoniae (100 μL, 10⁸ CCU). Cells were collected at 24 hpi, and subjected to western blotting to determine the expression of M. hyopneumoniae mhp384. **P < 0.01, and ***P < 0.001 indicate statistically significant differences among different groups.

Figure 5

Confocal laser scanning micrograph for the adhesion of M. hyopneumoniae cells to alveolar macrophages. Alveolar macrophages were treated with M. hyopneumoniae (A) and PBS (B), respectively. M. hyopneumoniae cells were labeled with CFDA-SE (green); cell nuclei were stained with DAPI (blue); and NOD1 was labeled red by using mouse anti-NOD1 mAb and goat anti-mouse IgG-Cy3. M. hyopneumoniae cells were adhered to the edges of alveolar macrophages, presenting a merged yellow signal, where M. hyopneumoniae was co-localized with the cellular NOD1 (white arrows). Scale bars = 5 µm.

Figure 6

mhp390 mediates M. hyopneumoniae adhesion to alveolar macrophages and interacts with NOD1. (A) Adhesion of rMhp390 to alveolar macrophages detected by flow cytometry. (B) The adhesion of M. hyopneumoniae to alveolar macrophages was inhibited by rMhp390. The cells were incubated with different concentrations of rMhp390 before infection. BSA (10 μg/mL) and 1 mL of RPMI 1640 alone were used as the negative and blank controls, respectively. (C) The adhesion of M. hyopneumoniae to alveolar macrophages was inhibited by anti-rMhp390 serum. M. hyopneumoniae was incubated with anti-rMhp390 serum diluted from 1:50 to 1:1000 before infection. The mixed negative serum (unimmunized rabbit serum, 1:50 dilution) was used as the negative control. ***P < 0.001 indicate statistically significant differences among different groups, and ns represents no difference. (D) Kinetic analysis of the affinity between mhp390 and NOD1 through bio-layer interferometry.

Figure 7

Schematic representation of the NOD1-RIP2 signaling pathway in M. hyopneumoniae invasion of alveolar macrophages.