If you’re over the age of 10, you’ve probably experienced the joys of having a pimple, and all the pain – physical and emotional – that goes along with it. But have you ever wondered why pimples hurt?
Typically we’ve assumed that the pain of an infection comes primarily from the inflammatory response your body produces to fight off the bacteria – cytokines, prostaglandins, and other mediators that activate nociceptors on pain-sensing neurons in your peripheral nervous system. Much like the misery of being feverish when you have the flu, this inflammatory pain is an unfortunate but necessary side effect of the immune response, protecting your body against invasion. But bacteria can be real jerks all on their own, and Dr. Clifford Woolf’s lab has uncovered evidence that some bacteria can directly activate nociceptors through N-formyl peptides and the pore-forming toxin a-haemolysin (aHL).
Dr. Clifford Woolf, of Harvard University, studies pain, regeneration and neurodegenerative diseases. Dr. Woolf uncovered the phenomenon of “central sensitization”, in which peripheral inflammation and tissue damage leads to sensitization of the nociceptive neurons in the dorsal horns of the spinal cord. This sensitization is mediated by NMDA receptors, and can be treated by opiates. His research on this subject is the driving force behind the current practice of treating pain early (for example, by giving morphine before surgery to preempt post-surgical sensitization). Dr. Woolf’s work has been key to better understanding mechanisms of human pain sensation, and plays an important role in the way that patient pain is treated in hospitals around the world.
In a recent study, his group looked directly at the molecular mechanisms of pain generation during Staphyloccocus aureus infection. Never heard of S. aureus? Maybe you know it as MRSA – that’s right, the antibiotic-resistant form of this bacteria is the bane of many hospitals.
Dr. Woolf’s group injected the hindpaws of mice with S. aureus and, as you might expect if someone injected your foot with a bunch of nasty bacteria, the mice showed mechanical, heat, and cold hypersensitivity within one hour. This lasted for 48-72 hours, with a peak at six hours after infection (Fig. 1a). By examining the kinetics of immune activation, they found that tissue swelling did not correlate with pain (Fig. 1a), and the influx of immune cells and cytokines increased in infected tissue but did not correlate with hyperalgesia (Fig. 1b, c). The bacterial load, however, showed a similar time course as that of pain hypersensitivity, peaking at 6 hours and decreasing over time as myeloid cells ingested the bacteria (Fig. 1d). This pain time course doesn’t quite match up if the inflammatory response is causing the pain – but it does match up with the presence of bacteria at the injury site.
The lab decided to examine whether key immune response pathways were necessary for S. aureus-induced pain using TLR2 and MyD88 knock-out mice, which removed the animal’s protection against S. aureus skin infection. The same mechanical and thermal hyperalgesia was seen, indicating that the pain response is not dependent on the immune activation. They also tried removing neutrophils and monocytes, important for immunity against the bacteria by limiting its survival and spread, by injecting a GR1 antibody before infection. This treatment resulted in an increase in mechanical and heat hypersensitivity, accompanied by a higher bacterial load. Finally, using NOD scid gamma (or, if you want to get extra-sciencey, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, they saw that knocking out natural killer T and B cells did not alleviate the acute bacterial pain. This seems to indicate that the pain sensation associated with S. aureus injection is not dependent on the immune response.
The strong correlation between pain and bacterial load led Dr. Woolf’s group to examine whether or not bacteria interact directly with nociceptors by applying heat-killed S. aureus to dorsal root ganglia (DRG) sensory neurons. This induced a calcium flux response and action potential firing in a subset of neurons that also respond to capsaicin (the chemical that makes spicy peppers “hurt so good” in your mouth) (Fig. 2a,b).
So how do the bacteria activate nociceptors? Woolf and his group targeted N-formylated peptides, bacterial molecules used by leukocytes to mediate immune chemotaxis during infection. Application of fMLF (E. coli-derived) and fMIFL (S. aureus-derived) both induced calcium flux in a subset of DRG neurons that also responded to capsaicin, similar to the response seen with bacterial application (Fig. 2e), and resulted in hyperalgesia when injected into mice (Fig. 2f).
FPR1 is the receptor that recognizes fMLF and fMIFL in immune cells, so the lab tried knocking it out in mice. Fpr1-/- mouse DRG neurons showed decreased calcium flux, and Fpr1-/- mice had reduced mechanical hyperalgesia after treatment with fMIFL relative to wild-type (Fig. 3g).
The lab also targeted aHL, a pore-forming toxin involved in tissue damage and bacterial spread. aHL can assemble pores in cell membranes allowing non-selective cation entry – which might be enough to depolarize cells. Like fMLF and fMIFL, aHL induced calcium flux in nociceptors on DRG neurons (Fig. 4a, b). When injected into mice, aHL induced pain behavior in a dose-dependent manner (Fig 4c). These effects did not involved voltage-gated calcium channels or large-pore cation channels, but did require external calcium – this would seem to indicate that the pores aHL assembles in the membrane are sufficient for depolarization. Knocking out aHL expression in S. aureus led to significantly less hyperalgesia than wild-type bacteria, indicating a robust role for aHL in pain during S. aureus infection.
Dr. Woolf’s group neatly summarized the mechanisms by which bacteria directly activate nociceptors in the diagram below:
Finally, the lab opted to ablate (remove) the nociceptive cells responsible for the S. aureus pain response to examine the role of nociceptors in modulating the immune response. Ablation of these cells led to significantly increased tissue swelling with increased infiltration of neutrophils and monocytes at the infection site and enlarged lymph nodes – indicating that nociceptor ablation led to increased local inflammation. This hints at a role for nociceptors directly modulating immune activation, and bacteria may be directly activating the nociceptors as a means to increase immunosuppression and reduce the ability of the host to clear the pathogen.
So not only do the bacteria directly activation your pain receptors, but they also might be making it harder for your body to fight them off. Makes those bacteria sound extra evil, doesn’t it? Think about that the next time you have a sore pimple – or, if you are blessed with good skin, the next time you have a gnarly hangnail.
Be sure to check out Dr. Clifford Woolf’s talk, “Studying human pain in a dish”, at 4 PM on Tuesday, April 29th in the CNCB Large Conference Room, if you’d like to hear more on this subject!
Alison Caldwell is a first year student in the UCSD Neurosciences Graduate Program. She is currently rotating under Dr. Chitra Mandyam studying the effects of addiction on neuronal proliferation and morphology in the hippocampus. She can be found on Twitter at @alie_astrocyte
Chiu I.M., Heesters B.A., Ghasemlou N., Von Hehn C.A., Zhao F., Tran J., Wainger B., Strominger A., Muralidharan S. & Horswill A.R. & (2013). Bacteria activate sensory neurons that modulate pain and inflammation, Nature, 501 (7465) 52-57. DOI: 10.1038/nature12479