Bacteria activate sensory neurons that modulate pain and inflammation

Abstract

Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.

Figure 1. S. aureus infection induces pain hypersensitivity paralleling bacterial load but not immune activation

(a) S. aureus infection induces mechanical hypersensitivity (p=0.0021, n= 10/group), heat hypersensitivity (p<0.0001, n=10/group), acetone cold response (p<0.0001, n=20/group), and tissue swelling (p<0.0001, n=10/group). *p<0.05, ***p<0.001. (b) Left, flow cytometry shows myeloid (CD11b⁺CD45⁺) but not lymphoid (CD11b⁻CD45⁺) immune expansion in infected tissues. Right, Quantification of infected tissue neutrophils (CD11b⁺Ly6G⁺), Ly6c^hi monocytes (CD11b⁺Ly6G⁻Ly6C^hi), and Ly6C^lo monocytes/macrophages (CD11b⁺Ly6G⁻Ly6C^lo). n=3/time-point. (c) TNF-α, IL-1β levels in infected tissues. n=4/time-point. (d) Bacterial load recovery. n=4/time-point. (e) GFP-S. aureus are in proximity with Nav1.8-Cre/TdTomato⁺ dermal nerve fibers, 3 hours post-infection. Scalebar,100 μm. Error bars, mean±s.e.m.

Figure 2. Innate immunity through TLR2/MyD88 and neutrophils/monocytes is not necessary for pain during S. aureus infection

(a) Infection-induced mechanical hypersensitivity is similar in TLR2^−/− mice (n=10 infected) compared to WT mice (n=10 infected, n=10 saline injected) (p=0.744), and MyD88^−/− mice (n=10 infected) relative to WT mice (n=11 infected, n=7 saline injected) (p=0.533). (b) Bacterial load, 3 days post-infection (n=5 each). (c) Infection-induced mechanical (p<0.0001) and heat (p<0.0001) hypersensitivity are increased in GR1 treated mice (n=10 infected, n=10 saline) compared to rat IgG treated mice (n=10 infected). Bonferroni, ***p<0.001. (d) Bacterial load, 2 days post-infection (n=6 each). Error bars, mean±s.e.m.

Figure 3. Bacterial heat-stable components including N-formylated peptides activate nociceptors

(a) Hk-S. aureus induces calcium flux in capsaicin, KCl responsive DRG neurons (arrows, traces). (b) (i) Representative recording, (ii) firing frequency upon hk-S. aureus application (5 capsaicin-responsive cells, 9 unresponsive). (c) DRG responsive proportions to hk-bacteria (n=4-26 fields/condition). (d) Acute pain induction: saline (n=13), hk-S. aureus (n=12), hk-S. pneumonia (n=14), hk-L. monocytogenes (n=5), hk-M. fermentans (n=6), hk-H. pylori (n=5), hk-P. aeruginosa (n=8), hk-E. coli (n=6). **p<0.01, *p<0.05. (e) DRG responsive proportions to formyl peptides (n=3-14 fields/condition). (f-g) fMLF, fMIFL injection induces mechanical hypersensitivity. Fpr1^−/− mice show reduced hk-S. aureus mechanical hypersensitivity (p=0.0089). fMIFL vs. saline, Fpr1^−/− vs. WT: *p<0.05; **p<0.01; ***p<0.001. Error bars, mean±s.e.m.

Figure 4. Heat-sensitive S. aureus Hla activates nociceptors and contributes to infection-induced hyperalgesia

(a) Hla application evoked DRG neuron calcium flux (arrows, traces), (b-c) dose-dependent calcium flux (n=3/condition) and acute pain (n=5-10/group). *p<0.05, ***p<0.001. (d) Heat pre-treatment abolishes Hla-induced pain (1 μg, n=7/group). (e) Hla, HLA_H35L evoked DRG neuron action potentials (arrow, Hla application, n=3/condition). (f) Hla (1 μg, n=6) but not Hla_H35L (1 μg, n=5) induced acute pain. (g) Hla 100 ng (n=8), 330 ng (n=8), saline (n=8) injection induced hypersensitivity. 100 ng vs. saline: ***p<0.001; 330 ng vs. saline: #p<0.05, ##p<0.01, ###p<0.001. (h) S. aureus lacking Hla (n=12) produced less mechanical (p=0.0056), heat (p=0.0193), acetone (p=0.0118) hypersensitivity than WT S. aureus (n=13). *p<0.05; **p<0.01; ***p<0.001. Error bars, mean±s.e.m.

Figure 5. Nociceptor ablation leads to increased local inflammation and lymphadenopathy following S. aureus infection

(a) Nav1.8-Cre/DTA neurons lack hk-bacteria responses. (b) Infection-induced mechanical (p=0.0027), heat hypersensitivity (p=0.0003) in Nav1.8-Cre/DTA mice (n=10 mechanical, n=6 heat) and Control littermates (n=12 mechanical, n=6 heat). ***p<0.001. (c-e) Parameters analyzed 24 hours post-infection. (c) Tissue swelling: Nav1.8-Cre/DTA (n=23), Control (n=19). (d) Plantar neutrophils/monocytes: Nav1.8-Cre/DTA: n=4 uninfected, n=15 infected; Control: n=4 uninfected, n=19 infected. (e) Popliteal lymph node images (infected), lymph node cellularity (Nav1.8-Cre/DTA: n=9 uninfected, n=10 infected; Control: n=9 uninfected, n=11 infected), and lymph node monocyte/macrophage (Mϕ), neutrophil (Nϕ), T, B cell subsets (n=5 each). Error bars, mean±s.e.m.

Figure 6. Nociceptor derived neuropeptides regulate innate immune activation

(a) Nav1.8-Cre/TdTomato⁺ DRG, trigeminal, nodose ganglia neurons were purified by flow cytometry (gates shown). (b) Top 30 nociceptor-expressed neuropeptides and myeloid immune cell-expressed neuropeptide receptors, shown from maximum to minimum. (c) Hla, S. aureus supernatant, and capsaicin (100 nM) induce DRG neuron CGRP release. **p<0.01; ***p<0.001. (d) TNF-α production by hk-S. aureus or Lipoteichoic acid stimulated macrophages was suppressed by CGRP, Sst, Gal (neuropeptide concentrations, 1 μM; *p<0.05). (e) CGRP injection decreased lymphadenopathy 24 hours post-S. aureus infection. Error bars, mean±s.e.m.