The Fight Against Bacterial Lung Infections: A New Role for Nociceptors?
The pain field is becoming increasingly aware of the links between the nervous and immune systems, and how complex interactions between the two underlie many pain disorders. A new study provides another interesting example of this crosstalk by highlighting the role that nociceptors can play in the immune response to infection. In particular, it describes a previously unrecognized role for these cells in regulating defense against bacterial lung infections and lethal pneumonia.
Research led by Isaac Chiu, Harvard Medical School, Boston, US, shows that TRPV1+ nociceptors regulate pulmonary host defense against lethal methicillin-resistant Staphylococcus aureus (MRSA) pneumonia by suppressing neutrophil and γδ T cell responses in mice. Selective ablation of TRPV1–positive neurons increased survival in these animals and promoted protective inflammatory cytokine production and clearance of the bacteria from the lungs. Immunosuppression by vagal ganglia TRPV1+ afferents depended upon release of calcitonin gene-related peptide (CGRP), suggesting that blocking CGRP signaling may represent an effective approach to improve host defense against bacterial lung infections and pneumonia.
“What is most striking about this paper is the somewhat unexpected role that nociceptors play in pulmonary host defense. It’s also intriguing how the role of nociceptors in host immunity differs depending on the type and even the site of infection. In pulmonary host defense the suppression of TRPV1 nociceptors is protective, but in other contexts it’s not,” said Marzia Malcangio, King’s College London, UK, who was not involved in the new study.
The findings were published online March 5 in Nature Medicine.
Unexpected neuroimmune crosstalk
Nociceptive neurons that innervate the respiratory tract are responsible for the coughing, constriction of airways (resulting in shortness of breath) and chest pain associated with pulmonary infections. It was long assumed that the pain associated with bacterial infections resulted from activation of nociceptors by local inflammation and by the immune cell response to the pathogens.
However, while working as a postdoc in Clifford Woolf’s lab at Boston Children’s Hospital and Harvard Medical School, Boston, US, Chiu discovered that bacteria could directly activate nociceptors in mice independently of local inflammation and the immune response. Perhaps more surprising was that ablation of nociceptors increased the immune response to the infection, suggesting that nociceptors may impair the activation of immune cells in response to bacterial stimulation (Chiu et al., 2013; see related PRF story here).
Intrigued by these findings, Pankaj Baral, first author of the current study and a trained immunologist with a background in bacterial infections (and who subsequently became a postdoc in Chiu’s lab), contacted Chiu. He asked him if he had considered neurons in the lungs and the role they may play in pulmonary infections.
“It was a great question,” said Chiu. “There are so many sensory fibers in the lungs, and they mediate important responses including pain and cough, but unknown was their role in host defense. As our background was in bacteria, we decided to concentrate primarily on bacterial infections.”
The researchers were interested in the role that TRPV1 nociceptors in particular may have in the response to pulmonary S. aureus (MRSA) infections, as previous work had shown that silencing these neurons reduced allergic inflammation in a mouse model of asthma (Talbot et al., 2015). So, in the current study, the group selectively ablated TRPV1 neurons in mice, either through a genetic approach (injection of diphtheria toxin into mice expressing the human diphtheria-toxin receptor under control of mouse TRPV1 regulatory sequences), or a chemical strategy (subcutaneous treatment with resiniferatoxin, a TRPV1 ligand).
Following a short recovery period after treatment with diphtheria toxin or resiniferatoxin, the animals were inoculated with a lethal dose of MRSA. While 80 percent of non-ablated control mice succumbed to MRSA infection, most TRPV1 neuron-ablated mice survived and showed improved maintenance of core body temperature as well as reduced bacterial burden in the lungs. Interestingly, ablation of nociceptive neurons expressing the voltage-gated sodium channel Nav1.8, which overlap with but are distinct from TRPV1 nociceptive neurons, produced significantly smaller beneficial effects compared to TRPV1 ablation.
A critical role for neutrophils
To address how the TRPV1 neurons regulated the host immune response, the team performed tissue staining of infected lungs in TRPV1 neuron-ablated and control mice. They saw higher immune cell influx into the lungs in TRPV1-ablated animals, alongside fewer bacterial colonies and earlier induction of proinflammatory cytokines, compared to control mice. This suggested a strong immune response to infection after TRPV1 ablation.
Looking more closely at the immune cells in lungs infected with S. aureus, the investigators discovered that TRPV1-ablated mice had more CD11b+ neutrophils, especially six and 12 hours after infection. Since neutrophils are critical to clear the lungs of bacteria, regulation of these immune cells by TRPV1 nociceptors could play a critical role in MRSA infection and pneumonia, the group reasoned.
Neutrophils account for the majority of white blood cells in the innate immune system in mammals, and their primary function is to fight infection. Within the lungs, neutrophils are constantly on the lookout for pathogens and display typical surveillance behaviors such as tethering (a transient interaction with the blood vessel wall), adhesion (a more advanced interaction with the vessel wall), and crawling (a complex intravascular behavior), each of which require increasing levels of cellular activation, respectively. Live intravital (in vivo) microscopy of the lungs revealed that neutrophils in TRPV1-ablated mice showed significantly more vascular crawling and greater crawling distances, both of which are consistent with host defense against bacterial pathogens.
To better understand which lung-resident immune cells in particular might be affected by TRPV1 neuron ablation, the researchers examined a range of immune cells including B cells, neutrophils, and γδ T cells. They discovered that ablated mice had higher numbers of γδ T cells in the lungs. So, the group used genetically engineered mice deficient in these cells, ablated all TRPV1 neurons, and then inoculated the animals with S. aureus. Mice lacking γδ T cells were no longer protected against bacterial infection and had no survival benefit after TRPV1 ablation, strongly suggesting that the contribution of nociceptors to host defense depended on γδ T cells.
“These γδ T cells were dysregulated even in uninfected TRPV1-ablated mice, suggesting that TRPV1 neurons were controlling their homeostasis,” explained Chiu.
In all of their ablation experiments so far, Chiu and his colleagues had removed all TRPV1 neurons. To learn if a subset of TRPV1 neurons was critical for the improved host defense and survival seen after ablation, they decided to target the vagus nerve, which is the major source of sensory input to the lung. The cell bodies of vagal afferents lie in the vagal ganglia at the base of the skull. To specifically target and ablate TRPV1 vagal afferents, they injected diphtheria toxin bilaterally to the vagal ganglia. Following a lethal dose of S. aureus, vagal TRPV1-ablated mice had a 90 percent survival rate compared to controls, all of which died. This increased survival also correlated with more neutrophils and a lower bacterial burden in the lungs.
The CGRP connection
Nociceptive neurons communicate with immune cells through the release of neuropeptides stored in nerve terminals. The team found increased levels of the neuropeptide CGRP in the lungs after S. aureus infection and lower levels in TRPV1-ablated mice. So they suspected that CGRP signaling from neurons to immune cells might play a role in MRSA pneumonia. In support of that idea, treatment with a CGRP antagonist in naïve (intact TRPV1) mice improved survival and core body temperature after S. aureus infection.
“That neuropeptides such as CGRP are involved in these sensory neuroimmune interactions makes sense in a way, as many immune cell types express receptors for neuropeptides released by neurons,” according to Chiu.
Could simply blocking CGRP signaling rather than ablating TRPV1 be enough to improve survival in MRSA pneumonia? Chiu doesn’t think it’s that simple.
“When you compare the survival benefits of TRPV1 neuron ablation to blocking CGRP signaling, neuronal ablation produces a much greater survival phenotype, almost 100 percent survival, while CGRP antagonism produces only about 40 percent. CGRP signaling may well be one of the mechanisms by which neurons suppress the immune system, but it’s most likely not the only one.”
Indeed, Malcangio wonders what other neuropeptides may play a role in this phenomenon. “It would be interesting to investigate the role of substance P, as it and CGRP can be both co-expressed and co-localized on TRPV1 neurons, and, one would assume, co-released. It could well be that in this context substance P does not have a major role and CGRP does, but it would be beneficial to know for sure.”
Different bacteria, different pathology
Interestingly, one finding from the study was that while TRPV1 or Nav1.8 ablation boosted the immune response to MSRA, each actually promoted the spread of bacteria from the lungs into the blood, which can be a major cause of sepsis (an overwhelming immune response to bacterial infection that can result in tissue damage, organ failure, and death). TRPV1 neurons, therefore, appear to have two distinct and somewhat opposing roles in pulmonary bacterial infections: suppressing the immune response but also aiding dissemination of bacteria in order to maintain integrity of the lung as a barrier surface.
Another intriguing finding was that mice infected with bacterial strains other than S. aureus did not show enhanced survival after TRPV1 neuron ablation, so Chiu wonders if the role of TRPV1 neurons in neuroimmune suppression may well be pathogen specific.
“The way that different bacteria induce pathology differs greatly. In the case of S. aureus, damage is caused primarily through secreted toxins that cause tissue necrosis, so you really need neutrophils to come in and kill bacteria, which is why these TRPV1 neurons are playing a big role in this local neuroimmune suppression,” he said.
Dara Bree is a postdoctoral fellow at Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, US.
Image credit: Patrick J. Lynch, medical illustrator (CC BY 2.5 [https://creativecommons.org/licenses/by/2.5]), via Wikimedia Commons.
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