Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway.

Chick embryos are a valuable model for studying immunity and vaccines. Therefore, it is crucial to investigate the molecular mechanism of the Mycoplasma gallisepticum (MG)-induced immune response in chick embryos for the prevention and control of MG. In this study, we screened for downregulated let-7d microRNA in MG-infected chicken embryonic lungs to explore its involvement in the innate immune mechanism against MG. Here, we demonstrated that low levels of let-7d are a protective mechanism for chicken embryo primary type II pneumocytes (CP-II) in the presence of MG. Specifically, we found that depressed levels of let-7 in CP-II cells reduced the adhesion capacity of MG. This suppressive effect was achieved through the activated mitogen-activated protein kinase phosphatase 1 (MKP1) target gene and the inactivated mitogen-activated protein kinase (MAPK) pathway. Furthermore, MG-induced hyperinflammation and cell death were both alleviated by downregulation of let-7d. In conclusion, chick embryos protect themselves against MG infection through the innate immune molecule let-7d, which may result from its function as an inhibitor of the MAPK pathway to effectively mitigate MG adhesion, the inflammatory response and cell apoptosis. This study may provide new insight into the development of vaccines against MG.

Mycoplasma gallisepticum escapes the host immune response via gga-miR-365-3p/SOCS5/STATs axis.

A disruption in the expression of gga-miR-365-3p was confirmed in the Mycoplasma gallisepticum (MG)-infected Chicken primary alveolar type II epithelial (CP-II) cells based on previous sequencing results, but the role it plays in the infection was unclear. In the present study, we demonstrate that MG evaded cellular host immunity via a gga-miR-365-3p/SOCS5-JAK/STATs negative feedback loop. Specifically, we found that at the initial stage of MG infection in cells, gga-miR-365-3p was rapidly increased and activated the JAK/STAT signaling pathway by inhibiting SOCS5, which induced the secretion of inflammatory factors and triggered immune response against MG infection. Over time, though, the infection progressed, MG gradually destroyed the immune defences of CP-II cells. In late stages of infection, MG escaped host immunity by reducing intracellular gga-miR-365-3p and inhibiting the JAK/STAT pathway to suppress the secretion of inflammatory factors and promote MG adhesion or invasion. These results revealed the game between MG and host cell interactions, providing a new perspective to gain insight into the pathogenic mechanisms of MG or other pathogens. Meanwhile, they also contributed to novel thoughts on the prevention and control of MG and other pathogenic infections, shedding light on the immune modulating response triggered by pathogen invasion and their molecular targeting.

DF-1 cells prevent MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis.

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed gga-miR-24-3p expression level was significantly increased in MG-infected chicken lungs. The aim of this study is to reveal the cellular mechanism behind the MG-HS infection. We found that gga-miR-24-3p was significantly upregulated and Ras-related protein-B (RAP1B) was downregulated in chicken fibroblast cells (DF-1) with MG infection. Dual luciferase reporting assay and rescue assay confirmed that RAP1B was the target gene of gga-miR-24-3p. Meanwhile, overexpressed gga-miR-24-3p increased the levels of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), and significantly inhibited cell proliferation as well as promoted MG-infected DF-1 cell apoptosis, whereas inhibition of gga-miR-24-3p had the opposite effect. More importantly, the results of overexpression and knockdown of target gene RAP1B demonstrated that the presence of RAP1B promoted cell proliferation and it saved the reduced or increased cell proliferation caused by overexpression or inhibition of gga-miR-24-3p. Furthermore, the overexpression of gga-miR-24-3p could significantly inhibit the expression of MG-HS adhesion protein. Taken together, these findings demonstrate that DF-1 cells can resist MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis, which provides a new mechanism of resistance to MG infection in vitro.

Down-regulated gga-miR-223 inhibits proliferation and induces apoptosis of MG-infected DF-1 cells by targeting FOXO3.

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed that gga-miR-223 expression level significantly decreased in MG-infected chicken lungs. The aim of this study was to reveal the role of gga-miR-223 in MG-induced CRD progression. We found that gga-miR-223 was remarkably down regulated and forkhead box O3 (FOXO3) was up-regulated in both MG-infected chicken embryos lungs and the chicken embryonic fibroblast cell line (DF-1) by qPCR. FOXO3 was verified as the target gene of gga-miR-223 through bioinformatics analysis and dual-luciferase reporter assay. Further studies showed that overexpressed gga-miR-223 could promote cell proliferation, cell cycle, and inhibit cell apoptosis by notably promoting the expression of cell cycle marker genes cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 6 (CDK6) and Cyclin D1 (CCND1) and inhibiting the expression of apoptosis markers Bcl-2-like 11(BIM), FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL). As expected, FOXO3 knockdown group got similar results. Overexpression of gga-miR-223 observably promoted cell multiplication, cell cycle progression, and inhibited apoptosis of MG-infected DF-1 cells, while inhibited gga-miR-223 had the opposite effect. Taken together, upon MG-infection, downregulated gga-miR-223 could decrease proliferation, cycle progression, and increase apoptosis through directly targeting FOXO3 to exert an aggravating MG-infectious effect.