Abstract
Mycobacterium avium subspecies paratuberculosis (MAP) persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10) upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs) and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a) expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2)-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage). ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2) are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis.
Highlights
Paratuberculosis or Johne’s disease is characterized by chronic granulomatous enteritis predominantly observed in ruminants
Our data suggest that downregulation of miR-27a in response to Mycobacterium avium subspecies paratuberculosis (MAP) strains and toll-like receptor 2 (TLR2) treatment shares a common response in both types of macrophages and indicate that the expression of miR-27a in MAP- and TLR2-treated cells may share a common signaling mechanism
After 6 h postinfection, we found a high level of pro-inflammatory cytokines, Figure 2 | Mycobacterium avium subspecies paratuberculosis (MAP) infection decreases miR-27a expression in macrophages and mice. (A,B) Bone marrowderived macrophage (BMDM) and (D,E) RAW264.7 cells were infected with MAP (k-10 and 0908) for the indicated time period, and miR-27a expression was subsequently evaluated by using qRT-PCR. (C) RAW264.7 and (F) Bone Marrow-Derived Macrophages (BMDMs) were stimulated with 1 μg/ml Pam3Cys-Ser-(Lys)4 [PAM toll-like receptor (TLR)1/2 agonist] for the indicated time period, and miR-27a expression was examined using qRT-PCR. (G–I) The expression levels of miR-27a were measured in the intestine (G), spleen (H), and liver (I) of negative control or MAP (0908)-infected C57BL/6 mice by qRT-PCR analysis
Summary
Paratuberculosis or Johne’s disease is characterized by chronic granulomatous enteritis predominantly observed in ruminants. It is caused by an obligate non-tuberculous Mycobacterium avium subspecies paratuberculosis (MAP), a member of M. avium complex. The USDA reports suggested an increasing trend of MAP infections in US dairy herds, from 21.6% in 1996 to 91.1% in 2007 [3]. A recent study from northeastern part of China reported that the prevalence of paratuberculosis is 4.8% at the animal level and 50.0% at the herd level [4]. Animals of all ages are susceptible to paratuberculosis, and the infection is acquired by fecal–oral route or ingestion of contaminated milk in young animals [5, 6]. In light of the current knowledge about MAP and its relationship with human disease, the majority of researchers support the theory that MAP causes CD in some genetically susceptible human hosts, but additional proof is needed for the confirmation of MAP as a causative agent of CD [10]
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