Net-like structures made of DNA and antimicrobial proteins protect the body from infection by trapping and containing large pathogens. New findings published in the journal Science show that a DNA repair protein is critical to keeping them stable and preventing them from triggering inflammation throughout the body.
NET structure with (left) and without the RAD51 protein (right). NETs without RAD51 were less dense and branched. Credit: Antimicrobial Defence Laboratory.
The immune system walks a fine line between protecting the body from infection and causing damage to healthy tissues. Crick group leader Veni Papayannopoulos is interested in how immune cells kill large pathogens like parasites and fungi, without stimulating widespread inflammation.
“Some pathogens are too large for immune cells to engulf, so the body relies on other mechanisms to keep them confined. One of the main ways immune cells called neutrophils capture pathogens are by secreting NETs, or ‘neutrophil extracellular traps.’ These are exactly what they sound like, biological webs that sit outside of the cell and trap and kill pathogens,” Veni explains.
But the activity of NETs requires delicate control. NETs do not only trap and kill pathogens. They are also strong inducers of inflammation, a process that recruits and activates immune cells to an area of the body that has been invaded by pathogens. Inflammation is beneficial when it is short and restricted to one area, but it can be detrimental when it is prolonged or when it spreads systemically.
Unexpectedly, Veni’s team addressed how inflammation is contained in one place by trying to address a fundamental question that had been neglected in the biology of NETs: how these complicated structures are built.
NETs are made of a type of genetic material called chromatin, a mixture of DNA and proteins which is normally tightly packaged inside the cell nucleus. In contrast, NET chromatin is large, loose and branched. Until now, the mechanisms that build this unique branched DNA network and its role in NET function remained unknown.
Veni explains that he’d long wondered what held together these bizarre branched structures, until a lecture at the Crick from group leader Steve West in 2017 pointed him in the right direction. “Steve, whose lab focuses on the process of DNA repair, showed a famous image of two interlocked DNA strands undergoing a repair process that involves RAD51, a protein that generates branched DNA intermediate structures,” says Veni. “I was struck that RAD51 and DNA repair might be the key to unlock the mechanism of NET branching. I reached out to Steve and he joked that if this were true, it might make the cover of Science.”
The researchers took images of NETs at a very high resolution, finding that RAD51 helps to form a robust branched web-like structure that holds NETs together and makes them more resistant to degradation. “We prevented cells from using RAD51, either with a drug or by turning off the gene that makes it, and the architecture of NETs changed,” PhD student and co-first author Sophie Guan says. “They had fewer branches and degraded more easily.”
Pulmonary aspergillosis is a group of inflammatory lung conditions caused by breathing in Aspergillus mould spores, often affecting people with conditions such as asthma. Since NETs can fuel inflammation, the team wondered whether disrupting their formation might reduce the severity of the disease. Instead, they found the opposite.
“We blocked RAD51 in mice infected with Aspergillus,” says Lorenza Iolanda Tsansizi, a principal laboratory research scientist in the lab and co-first author. “This led to an abnormal immune response, where fragments of NETs built up in the bloodstream. This in turn stimulated production of a pro-inflammatory molecule called IL-6 by activating immune cells in the bloodstream called monocytes.”
The increase in IL-6 changed the immune response in the mice. They produced more mucus, which narrowed their airways, and had higher numbers of immune cells called eosinophils. “We were seeing an exacerbated form of what we call type-2 inflammation,” says Sophie. “This is where the whole body responds. It’s commonly associated with a misfired immune response to harmless substances like pollen or dust, leading to allergies, severe asthma or hayfever.”
The team then looked at data from people with different types of pulmonary aspergillosis. Those who had NET fragments in their blood also had higher levels of pro-inflammatory molecules like IL-6 and indicators of higher eosinophil activity. Although the team did not clarify whether NET instability was the primary cause of cell free chromatin accumulation in the bloodstream of patients, there was a strong correlation between NETs, IL6 and eosinophil markers that supports a role for this dysregulated inflammatory pathology.
The collaboration between the two labs demonstrates how answers might come from taking a multidisciplinary view of biology. “That RAD51 promotes NET formation is a remarkable and unexpected observation by Veni’s lab,” says Steve. “We were only too happy to provide a little expertise regarding RAD51, to help connect the two seemingly disparate fields of biological research.”
For Veni, the project is proof that NET architecture is intrinsically linked to their functions, with some surprising findings. “We didn’t expect that destabilising NETs would result in systemic inflammation by an unexpected pathway,” he concludes. “It’s clear that NETs need to be deployed in a way that restricts them to one location, and we anticipate that defects in NET stability may help us understand mechanisms behind a range of diseases.”
