Respiratory infections of the upper and lower airways trigger a range of symptoms, including altered breathing, social disengagement, malaise and lethargy, which result from the systemic inflammatory response to infection. Various microorganisms can lead to respiratory infections. An example is the Gram-negative bacterium Pseudomonas aeruginosa, which can cause both acute and chronic lung infections. Typically, P. aeruginosa resides within biofilms that pose a challenge for treatment, as they protect the bacteria from antibiotics and the host immune response. P. aeruginosa biofilms are formed mainly by exopolysaccharide (EPS) molecules like Pel and Psl. The impact of EPS on host immunity and disease in vivo remains unknown. In this study, Granton et al. link the symptoms and behavioural changes presented during P. aeruginosa lung infection with biofilm formation, showing that biofilm-associated EPS controls sickness.

First, the authors used two engineered strains of P. aeruginosa PAO1 to assess the impact of EPS production on host responses and sickness. One strain produced EPS (EPS+), whereas the other did not (EPS–). The authors examined the phenotypes of these strains (the EPS+ strain aggregated whereas the EPS– strain did not) and then demonstrated their effects in mice. Mice infected with the EPS– strain developed high-grade sickness, whereas those infected with the EPS+ strain developed low-grade sickness. EPS– infections also resulted in more severe alterations in the activity and behaviour of mice, including avoidance of external stimuli, and reduced exploration and searching. EPS–-infected mice also developed worse hypothermia than EPS+-infected mice. However, the relationship between EPS production and sickness was not so simple; EPS+ infections in neutropenic mice were lethal, whereas EPS– infection was not, which suggests a multifaceted link between infectious state, sickness, neutrophil host defence and inflammation.

Credit: Philip Patenall/Springer Nature Limited

Next, the authors hypothesized that EPS could help bacteria evade host detection of lipopolysaccharide (LPS), a major pathogen-associated molecular pattern of Gram-negative bacteria that binds to host Toll-like receptor 4 (TLR4). Indeed, by infecting Tlr4–/– mice with P. aeruginosa EPS+ and EPS– strains, the researchers demonstrated that EPS molecules hide LPS from being recognized by TLR4 during P. aeruginosa infection, resulting in impaired clinical sickness. This was also evidenced by attenuated sickness and hypothermia in TLR4-deficient mice infected with the EPS– strain.

So, what is the underlying signalling pathway accounting for this observation? The authors decided to focus on sensory neurons, also called nociceptors, as they have been shown to modulate the infection outcome during Staphylococcus aureus pneumonia. Upon infection, TRPV1+ nociceptors are activated by pathogenic factors and release immune-altering neuropeptides, while also sending signals to the brain. The authors created a Tlr4-knockout mouse only in TRPV1+ sensory neurons and demonstrated that TRPV1+ lung sensory neurons mediate sickness through the LPS–TLR4 pathway during P. aeruginosa pneumonia.

To investigate whether sickness involves lung sensory neuron activation and communication with the central nervous system, the authors performed a series of in vivo experiments and demonstrated that the detection of LPS by TLR4 of lung TRPV1+ sensory neurons mediates a stress response in the paraventricular nuclei of the hypothalamus by activating corticotropin-releasing hormone neurons responsible for sickness behaviour and hypothermia. Such a mechanism was diminished in EPS+ infections.

“biofilm-associated EPS controls sickness”

In sum, this study demonstrates a mechanism by which sickness is dependent on P. aeruginosa EPS production.