Thursday, July 14, 2016

Are NETs involved in fighting Leptospira interrogans infections?

Neutrophils are the most abundant white blood cells in the bloodstream.  As the first immune cells to be recruited to infected tissues, they play a key role in the fighting microbial intruders.  It's long been known that they engulf microbes by phagocytosis, which results in the microbes being imprisoned within phagosomes inside the neutrophil.  Deadly proteases, antimicrobial proteins, and reactive oxygen species are released into the phagosome to kill the microbes.

Another means used by neutrophils to kill microbes was discovered just a decade ago.  When mixed with bacteria, neutrophils cast nets of DNA impregnated with antimicrobial proteins to trap and kill the bacteria.  The web-like DNA goes by the name "neutrophil extracellular trap" (NET).  Several bacteria are known to trigger neutrophils to cast NETs, and NETs have even been observed by microscopy within infected tissues.

Fluorescence staining of a neutrophil exudate in an appendicitis case.  NETs are the fibrous material.  Figure 4H from Brinkmann et al., 2004.  Bar = 50 μm.
A study published last year in PLOS NTD showed that the spirochete Leptospira interrogans is also killed by NETs.  The image below shows the spirochetes trapped in a NET cast by a human neutrophil.

Human neutrophils were cultured with L. interrogans for 3 hours.  Figure 1A from Scharrig et al., 2015.  Bar = 50 μm.
The real question is whether NETs are involved in killing L. interrogans during infection.  To answer this question, the investigators turned to the mouse model of leptospirosis.  They found that the number of spirochetes in the bloodstream more than doubled when the neutrophils in the mice were depleted by injection of a monoclonal antibody targeting a antigen located on the neutrophil surface.  Later in the infection, there was 10-fold more spirochetes in the kidneys of mice whose neutrophils were depleted than in those with normal numbers of neutrophils.  This confirmed that neutrophils were involved in limiting infections by L. interrogans, but did the neutrophils fight the infection by casting NETs?

The investigators used an indirect method to measure the amount of NETs generated during infection.  Neutrophils often expel nuclear DNA in the form of nucleosomes to generate NETs.  (Nucleosomes are assembled by wrapping nuclear DNA around histones.)  For this reason, the investigators measured the levels of free nucleosomes in the bloodstream of infected mice by ELISA. They concluded that NETs were generated by neutrophils in the bloodstream because they detected free nucleosomes in blood drawn from infected mice.  Much less was detected when neutrophils were first depleted with the anti-neutrophil antibody, confirming that the main source of free nucleosomes was neutrophils.

These results don't convince me that NETs are generated by neutrophils during L. interrogans infection.  There could be other reasons for free nucleosomes being present in the bloodstream.  For example, nucleosomes could be released from neutrophils simply dying from their battle against L. interrogans.  More convincing evidence would be direct observation of NETs in infected animals, as done in this study of mice with E. coli blood infections.


Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, & Gómez RM (2015). Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. PLoS Neglected Tropical Diseases, 9 (7) PMID: 26161745

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, & Zychlinsky A (2004). Neutrophil extracellular traps kill bacteria. Science (New York, N.Y.), 303 (5663), 1532-5 PMID: 15001782