Scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA) bacteria being engulfed by a neutrophil [National Institute of Allergy and Infectious Diseases, National Institutes of Health]

Research has found that, similar to a spider trapping its prey, the immune system’s neutrophil and macrophage cells can cooperate to capture and “eat” bacteria. Researchers at the Vanderbilt Institute for Infection, Immunology, and Inflammation, hope that the newly identified antibacterial mechanism could inspire novel anti-virulence strategies against Staphylococcus aureus and other extracellular bacterial pathogens.

It was known that neutrophils—first responder immune cells that migrate to sites of infection—can self-destruct and release their protein and DNA contents to generate neutrophil extracellular traps (NETs). The new research, headed by postdoctoral fellow Andrew Monteith, PhD, has found that NETs boost the bacterial killing power of macrophages. “Neutrophils produce the spider webs that immobilize the bacteria, and macrophages are the spiders that engulf and kill the bacteria,” said Eric Skaar, PhD, the Ernest W. Goodpasture professor of pathology, microbiology, and immunology and director of the Vanderbilt Institute for Infection, Immunology, and Inflammation. Skaar is corresponding author of the team’s published research in Science Advances, which is titled, “Neutrophil extracellular traps enhance macrophage killing of bacterial pathogens.”

Staphylococcus aureus (“staph”) bacteria—particularly antibiotic-resistant forms—are a leading cause of hospital-acquired infections, infectious heart disease, and pus-forming skin and soft tissue infections. The gram-positive bacterium is the second leading cause of bloodstream infections, the authors noted.

Neutrophils and macrophages are both phagocytic cells known for ingesting bacteria and producing antimicrobial peptides (AMPs), reactive oxygen species, and other enzymes to fight infection. NET generation (NETosis), thought to be a form of programmed cell death, is a relatively recently discovered neutrophil antibacterial strategy, Skaar said. The released neutrophil DNA creates a sticky trap that is also studded with antimicrobial peptides.

“Professional phagocytes, such as neutrophils and macrophages (Mφs), have an arsenal of antimicrobial processes that are critical to combating bacterial pathogens,” the team wrote. “During infection, phagocytosis of S. aureus by neutrophils initiates bacterial killing through the production of reactive oxygen species (ROS) … In addition, neutrophils combat extracellular pathogens by secreting NETs consisting of DNA studded with AMPs through a process termed NETosis.”

Macrophages are also critical to the innate immune response to S. aureus, the team added, and elicit most of their antimicrobial activity after internalizing the pathogen. “Following phagocytosis, Mφs generate ROS and reactive nitrogen species, mobilize transition metals, and acidify the phagosome to activate hydrolytic enzymes in an effort to intoxicate pathogens,” they explained. However, despite these antimicrobial functions, “Mφs are not effective at eradicating internalized S. aureus in isolation,” they continued. And while its known that neutrophils can synergistically enhance the antibacterial function of macrophages, most host pathogen interactions studies involving S. aureus have been assessed using isolated immune cells or whole blood lacking mature Mφs. “Defining how neutrophils and Mφs cooperatively combat S. aureus will help define an effective innate immune response to S. aureus and to provide insight into antibacterial strategies that could be broadly applied to other extracellular bacterial pathogens.”

For their newly reported studies, Monteith and colleagues used neutrophils that undergo increased NETosis in animal and in vitro model systems, to study the biological function of NETs. They found that increased NETosis did not provide a killing advantage to neutrophils in isolation. But when macrophages were present, NET formation enhanced macrophage antibacterial activity by increasing phagocytosis—of staph bacteria stuck in the NETs along with neutrophil antimicrobial peptides. “The macrophages end up with not only their own antibacterial arsenal, but also the neutrophils’ antibacterial arsenal, all in the same compartment killing the bacteria,” Skaar said.

Increased NETosis also boosted macrophage killing of other bacterial pathogens, suggesting that neutrophil/NET-macrophage cooperation may represent a broadly applied immune defense mechanism. “Similar results were observed in response to other phylogenetically distinct bacterial pathogens including Streptococcus pneumoniae and Pseudomonas aeruginosa, implicating this as an immune defense mechanism that broadly enhances antibacterial activity,” the authors stated.

The researchers also showed that elimination of a staph nuclease enzyme that cuts DNA makes the bacteria even more sensitive to NET-macrophage killing. “It seems as if extracellular pathogens like staph have evolved secreted nucleases so they can cut their way out of these NETs—chop off the spider web and escape,” Skaar said.

Blocking the nuclease would make the pathogens more susceptible to NET-mediated killing and may be a good antibacterial treatment strategy. This type of “anti-virulence” approach would allow phagocytic and other immune cells to do their jobs and kill the bacteria, Skaar further suggested.

“Our results demonstrate that NET formation acts as a conduit to broadly enhance antimicrobial activity in the presence of Mφs in response to bacterial pathogens,” the authors concluded. “NET formation increases the antibacterial activity of Mφs by facilitating their phagocytosis of bacteria and by transferring biologically active neutrophil-specific AMPs to Mφs … These results demonstrate that achieving maximal bactericidal activity through NET formation requires macrophages and that accelerated and more robust suicidal NETosis makes neutrophils adept at increasing antibacterial activity…”

“Scientists are excited about the idea of anti-virulence strategies, because we know a lot about bacterial virulence mechanisms and can come up with creative ways to inhibit them,” Skaar commented. Current pharmaceutical efforts, however, focus on drugs that directly kill bacteria rather than blunting their virulence.

Monteith, Skaar, and colleagues are continuing to explore questions of NETosis, including how and when neutrophils opt for this form of cell death. They are also interested in how individual differences in NETosis—perhaps because of genetic variation or disease states—affect infection. In people with certain autoimmune conditions, for example, reduced NETosis may increase susceptibility to staph infections.

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