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Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira.

Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, Gómez RM - PLoS Negl Trop Dis (2015)

Bottom Line: Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA.Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice.Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Viruses, Institute of Biotechnology and Molecular Biology, CCT-La Plata, CONICET-UNLP, Buenos Aires, Argentina.

ABSTRACT
NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.

No MeSH data available.


Related in: MedlinePlus

Leptospira interrogans induce NETs in a concentration-dependent manner.Human neutrophils (2x105/mL) were incubated with Leptospira interrogans serovar Copenhageni (LIC) (MOI = 50) for 180 min and (A) visualized with a Nikon E200 photomicroscope or (B) fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (C) DNA or nucleosomes were measured by fluorometry (black bars) or ELISA kit (white bars) respectively in supernatants of LICa (bacteria alone) or unstimulated neutrophils (None) (500/μL) (negative control), stimulated with PMA (50 ng/mL) (positive control), or with LIC (MOI 50, 5 or 1) for 180 min Bars represent standard error of the mean (SEM) of assays from 3–10 independent assays; ***p <0.0001 vs. None. ##p<0.01 and ####p<0.0001 vs. LIC 50.
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pntd.0003927.g001: Leptospira interrogans induce NETs in a concentration-dependent manner.Human neutrophils (2x105/mL) were incubated with Leptospira interrogans serovar Copenhageni (LIC) (MOI = 50) for 180 min and (A) visualized with a Nikon E200 photomicroscope or (B) fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (C) DNA or nucleosomes were measured by fluorometry (black bars) or ELISA kit (white bars) respectively in supernatants of LICa (bacteria alone) or unstimulated neutrophils (None) (500/μL) (negative control), stimulated with PMA (50 ng/mL) (positive control), or with LIC (MOI 50, 5 or 1) for 180 min Bars represent standard error of the mean (SEM) of assays from 3–10 independent assays; ***p <0.0001 vs. None. ##p<0.01 and ####p<0.0001 vs. LIC 50.

Mentions: In order to investigate whether Leptospira sp. was able to induce NETs, we performed a series of initial experiments using Leptospira interrogans serovar Copenhageni (LIC) and human neutrophils. Stimulation of neutrophils with LIC resulted in the release of fine strands of extracellular DNA with bacteria trapped at their ends (Fig 1A). Using PMA as a positive control, the generation of NETs was confirmed by double-labeling studies with PI, to identify DNA, and with an antibody against elastase, a major neutrophil granular-derived protein that decorates the DNA strands (Fig 1B). Quantitative assays of the released DNA or nucleosomes indicated that the formation of NETs was dependent on bacterial concentration (Fig 1C). DNA levels in supernatants of LIC alone (without neutrophils) were not significant (Fig 1C).


Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira.

Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, Gómez RM - PLoS Negl Trop Dis (2015)

Leptospira interrogans induce NETs in a concentration-dependent manner.Human neutrophils (2x105/mL) were incubated with Leptospira interrogans serovar Copenhageni (LIC) (MOI = 50) for 180 min and (A) visualized with a Nikon E200 photomicroscope or (B) fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (C) DNA or nucleosomes were measured by fluorometry (black bars) or ELISA kit (white bars) respectively in supernatants of LICa (bacteria alone) or unstimulated neutrophils (None) (500/μL) (negative control), stimulated with PMA (50 ng/mL) (positive control), or with LIC (MOI 50, 5 or 1) for 180 min Bars represent standard error of the mean (SEM) of assays from 3–10 independent assays; ***p <0.0001 vs. None. ##p<0.01 and ####p<0.0001 vs. LIC 50.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498591&req=5

pntd.0003927.g001: Leptospira interrogans induce NETs in a concentration-dependent manner.Human neutrophils (2x105/mL) were incubated with Leptospira interrogans serovar Copenhageni (LIC) (MOI = 50) for 180 min and (A) visualized with a Nikon E200 photomicroscope or (B) fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (C) DNA or nucleosomes were measured by fluorometry (black bars) or ELISA kit (white bars) respectively in supernatants of LICa (bacteria alone) or unstimulated neutrophils (None) (500/μL) (negative control), stimulated with PMA (50 ng/mL) (positive control), or with LIC (MOI 50, 5 or 1) for 180 min Bars represent standard error of the mean (SEM) of assays from 3–10 independent assays; ***p <0.0001 vs. None. ##p<0.01 and ####p<0.0001 vs. LIC 50.
Mentions: In order to investigate whether Leptospira sp. was able to induce NETs, we performed a series of initial experiments using Leptospira interrogans serovar Copenhageni (LIC) and human neutrophils. Stimulation of neutrophils with LIC resulted in the release of fine strands of extracellular DNA with bacteria trapped at their ends (Fig 1A). Using PMA as a positive control, the generation of NETs was confirmed by double-labeling studies with PI, to identify DNA, and with an antibody against elastase, a major neutrophil granular-derived protein that decorates the DNA strands (Fig 1B). Quantitative assays of the released DNA or nucleosomes indicated that the formation of NETs was dependent on bacterial concentration (Fig 1C). DNA levels in supernatants of LIC alone (without neutrophils) were not significant (Fig 1C).

Bottom Line: Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA.Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice.Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Viruses, Institute of Biotechnology and Molecular Biology, CCT-La Plata, CONICET-UNLP, Buenos Aires, Argentina.

ABSTRACT
NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.

No MeSH data available.


Related in: MedlinePlus