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Antagonism of miR-328 increases the antimicrobial function of macrophages and neutrophils and rapid clearance of non-typeable Haemophilus influenzae (NTHi) from infected lung.

Tay HL, Kaiko GE, Plank M, Li J, Maltby S, Essilfie AT, Jarnicki A, Yang M, Mattes J, Hansbro PM, Foster PS - PLoS Pathog. (2015)

Bottom Line: Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses.Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity.Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.

View Article: PubMed Central - PubMed

Affiliation: Priority Research Centre for Asthma and Respiratory Disease, Department of Microbiology and Immunology, School of Pharmacy and Biomedical Sciences, Faculty of Health and Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia.

ABSTRACT
Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.

No MeSH data available.


Related in: MedlinePlus

Inhibiting miR-328 improves NTHi clearance in the lungs in vivo.MiR-328 was inhibited in (A) macrophages or (C) neutrophils for 12 h ex vivo. Then macrophages or neutrophils were labelled with CFSE and adoptively transferred i.t. into naïve mice, which were then infected with NTHi according to the timelines depicted. (E) Mice were inoculated with NTHi and 6 h later ant-328 or scrambled antagomir was administered to the lungs i.t. After 12 h (F) miR-328 expression was determined using Taqman PCR, normalised to sno-202 and expressed as fold change compared to the scrambled antagomir treatment. (B,D,G) Bacterial load in the lungs was measured by plating and counting bacterial colonies from lung homogenates in each model, and (H) BAL fluid was collected, cells isolated by cytospin and stained, and the total cellular infiltrate was assessed. Results are expressed as mean ± SEM (n = 6–8 mice per group; * p<0.05, ** p<0.01, *** p<0.001 vs scrambled antagomir control).
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ppat.1004549.g003: Inhibiting miR-328 improves NTHi clearance in the lungs in vivo.MiR-328 was inhibited in (A) macrophages or (C) neutrophils for 12 h ex vivo. Then macrophages or neutrophils were labelled with CFSE and adoptively transferred i.t. into naïve mice, which were then infected with NTHi according to the timelines depicted. (E) Mice were inoculated with NTHi and 6 h later ant-328 or scrambled antagomir was administered to the lungs i.t. After 12 h (F) miR-328 expression was determined using Taqman PCR, normalised to sno-202 and expressed as fold change compared to the scrambled antagomir treatment. (B,D,G) Bacterial load in the lungs was measured by plating and counting bacterial colonies from lung homogenates in each model, and (H) BAL fluid was collected, cells isolated by cytospin and stained, and the total cellular infiltrate was assessed. Results are expressed as mean ± SEM (n = 6–8 mice per group; * p<0.05, ** p<0.01, *** p<0.001 vs scrambled antagomir control).

Mentions: To specifically assess the role of miR-328 in macrophage- and neutrophil-mediated bacterial clearance in vivo we conducted adoptive transfer experiments. Macrophages or neutrophils were isolated from naïve mice and then treated with ant-328 or Scr control for 12 h before i.t. transfer into recipient naïve BALB/c mice (Fig 3A and 3C). To monitor the effectiveness of transfer, cells were pre-stained with CFSE. Mice were then inoculated with NTHi. Both CFSE-labelled macrophages (S12B and S12C Fig) and neutrophils (S12E and S12F Fig) entered the lungs and remained there for at least 24 and 2 h, respectively. There were no differences in the numbers of these cells between ant-328- and Scr-treated controls. Total bacterial load in BAL fluid and lung homogenates was measured by plating and colony counting. Importantly, mice that received ant-328 treated macrophages (Fig 3A and 3B) or neutrophils (Fig 3C and 3D) showed significantly improved clearance of NTHi from the lungs compared to Scr control treatment. However, there were no statistical differences in total inflammatory cell infiltrates in BAL fluid (S13A and S13F Fig). Mice that received ant-328 treated macrophages had similar total number of macrophages (S13B Fig) but a significantly reduced number of neutrophils (S13C Fig). It is likely that the reduction in neutrophil numbers is due to increased clearance of bacteria by ant-328 which leads to reduction in the early inflammatory events that promote neutrophil influx. The adoptive transfer of macrophages is likely to promote the removal of apoptotic neutrophils as well. Mice that received ant-328 treated neutrophils had no difference in macrophages (S13G Fig) or neutrophils numbers (S13H Fig). The concentrations of pro-inflammatory cytokines, IL-6 and TNF-α, in BAL fluid were equivalent in all experiments (S13D–S13E and S13I–S13J Fig). These data suggest that the effect of ant-328 on bacterial clearance was mediated directly by phagocytes and not by increased inflammatory cell recruitment or cytokine production.


Antagonism of miR-328 increases the antimicrobial function of macrophages and neutrophils and rapid clearance of non-typeable Haemophilus influenzae (NTHi) from infected lung.

Tay HL, Kaiko GE, Plank M, Li J, Maltby S, Essilfie AT, Jarnicki A, Yang M, Mattes J, Hansbro PM, Foster PS - PLoS Pathog. (2015)

Inhibiting miR-328 improves NTHi clearance in the lungs in vivo.MiR-328 was inhibited in (A) macrophages or (C) neutrophils for 12 h ex vivo. Then macrophages or neutrophils were labelled with CFSE and adoptively transferred i.t. into naïve mice, which were then infected with NTHi according to the timelines depicted. (E) Mice were inoculated with NTHi and 6 h later ant-328 or scrambled antagomir was administered to the lungs i.t. After 12 h (F) miR-328 expression was determined using Taqman PCR, normalised to sno-202 and expressed as fold change compared to the scrambled antagomir treatment. (B,D,G) Bacterial load in the lungs was measured by plating and counting bacterial colonies from lung homogenates in each model, and (H) BAL fluid was collected, cells isolated by cytospin and stained, and the total cellular infiltrate was assessed. Results are expressed as mean ± SEM (n = 6–8 mice per group; * p<0.05, ** p<0.01, *** p<0.001 vs scrambled antagomir control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4404141&req=5

ppat.1004549.g003: Inhibiting miR-328 improves NTHi clearance in the lungs in vivo.MiR-328 was inhibited in (A) macrophages or (C) neutrophils for 12 h ex vivo. Then macrophages or neutrophils were labelled with CFSE and adoptively transferred i.t. into naïve mice, which were then infected with NTHi according to the timelines depicted. (E) Mice were inoculated with NTHi and 6 h later ant-328 or scrambled antagomir was administered to the lungs i.t. After 12 h (F) miR-328 expression was determined using Taqman PCR, normalised to sno-202 and expressed as fold change compared to the scrambled antagomir treatment. (B,D,G) Bacterial load in the lungs was measured by plating and counting bacterial colonies from lung homogenates in each model, and (H) BAL fluid was collected, cells isolated by cytospin and stained, and the total cellular infiltrate was assessed. Results are expressed as mean ± SEM (n = 6–8 mice per group; * p<0.05, ** p<0.01, *** p<0.001 vs scrambled antagomir control).
Mentions: To specifically assess the role of miR-328 in macrophage- and neutrophil-mediated bacterial clearance in vivo we conducted adoptive transfer experiments. Macrophages or neutrophils were isolated from naïve mice and then treated with ant-328 or Scr control for 12 h before i.t. transfer into recipient naïve BALB/c mice (Fig 3A and 3C). To monitor the effectiveness of transfer, cells were pre-stained with CFSE. Mice were then inoculated with NTHi. Both CFSE-labelled macrophages (S12B and S12C Fig) and neutrophils (S12E and S12F Fig) entered the lungs and remained there for at least 24 and 2 h, respectively. There were no differences in the numbers of these cells between ant-328- and Scr-treated controls. Total bacterial load in BAL fluid and lung homogenates was measured by plating and colony counting. Importantly, mice that received ant-328 treated macrophages (Fig 3A and 3B) or neutrophils (Fig 3C and 3D) showed significantly improved clearance of NTHi from the lungs compared to Scr control treatment. However, there were no statistical differences in total inflammatory cell infiltrates in BAL fluid (S13A and S13F Fig). Mice that received ant-328 treated macrophages had similar total number of macrophages (S13B Fig) but a significantly reduced number of neutrophils (S13C Fig). It is likely that the reduction in neutrophil numbers is due to increased clearance of bacteria by ant-328 which leads to reduction in the early inflammatory events that promote neutrophil influx. The adoptive transfer of macrophages is likely to promote the removal of apoptotic neutrophils as well. Mice that received ant-328 treated neutrophils had no difference in macrophages (S13G Fig) or neutrophils numbers (S13H Fig). The concentrations of pro-inflammatory cytokines, IL-6 and TNF-α, in BAL fluid were equivalent in all experiments (S13D–S13E and S13I–S13J Fig). These data suggest that the effect of ant-328 on bacterial clearance was mediated directly by phagocytes and not by increased inflammatory cell recruitment or cytokine production.

Bottom Line: Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses.Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity.Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.

View Article: PubMed Central - PubMed

Affiliation: Priority Research Centre for Asthma and Respiratory Disease, Department of Microbiology and Immunology, School of Pharmacy and Biomedical Sciences, Faculty of Health and Hunter Medical Research Institute, University of Newcastle, Newcastle, Australia.

ABSTRACT
Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.

No MeSH data available.


Related in: MedlinePlus