TRPV1+ neurons promote cutaneous immunity against Schistosoma mansoni

Abstract

Immunity against skin-invasive pathogens requires mechanisms that rapidly detect, repel, or immobilize the infectious agent. While bacteria often cause painful cutaneous reactions, host skin invasion by the human parasitic helminth Schistosoma mansoni often goes unnoticed. This study interrogated whether pain-sensing skin afferents marked by expression of the ion channel Transient Receptor Potential Vanilloid 1 (TRPV1) contributed to the detection and initiation of skin immunity against S. mansoni. Data show that percutaneous S. mansoni infection significantly reduced thermal pain sensitivity evoked by TRPV1+ neurons. Consistently, isolated skin sensory neurons from infected mice had significantly reduced calcium influx and neuropeptide release in response to the TRPV1 agonist capsaicin compared to neurons from naïve controls. Using gain- and loss-of-function approaches to test whether TRPV1+ neurons initiate host-protective responses revealed that TRPV1+ neurons limit S. mansoni skin entry and migration into the pulmonary tract. Moreover, TRPV1+ neurons were both necessary and sufficient to promote proliferation and cytokine production from dermal γδ T cells and CD4+ T helper cells, as well as to enhance neutrophil and monocyte recruitment to the skin. Taken together, this work suggests that S. mansoni may have evolved to manipulate TRPV1+ neuron activation as a countermeasure to limit IL-17-mediated inflammation, facilitating systemic dissemination and chronic parasitism.

Keywords: T cells; neuroimmunology; neutrophils; parasitic-helminth; skin.

Graphical abstract

Figure 1.

Schistosoma mansoni infection reduces TRPV1+ neuron responses. (A) Experimental approach for percutaneous exposure to S. mansoni in the paw followed by assessment of thermal pain sensitivity 1 d after infection and DRG collection 2 d post-infection. DRG cultures were subsequently evaluated for capsaicin-induced calcium influx and neuropeptide release. (B) Thermal pain sensitivity in the ipsilateral (exposed) and contralateral (unexposed) paws of naïve and S. mansoni infected mice assessed by withdrawal times (seconds) to infrared heat. (C, D) Representative image of calcium traces of DRG neuron cultures generated from wild-type naïve or 2-d S. mansoni-infected mice. DRG neurons were sequentially treated with 1 μM capsaicin for 5 minutes followed by stimulation with 70 mM KCl for 1 minute. (E), Percentage of capsaicin-responsive DRG neurons per coverslip in naïve and S. mansoni-infected mice. (F) Area under the curve (AUC) values of intracellular calcium levels of wildtype DRG neurons from naïve or S. mansoni-infected mice during capsaicin treatment. (G, H), CGRP and Substance P supernatant levels of wildtype DRG neurons generated from wildtype naïve, or 2-d S. mansoni-infected ipsilateral and contralateral paws treated with vehicle or 1 μM capsaicin for 1 h. P values were determined by 2-tailed Student t-tests, or One-way ANOVA with post hoc correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative of 2-3 independent experiments, each with ≥4 biological replicates. Panel A was created in Biorender.

Figure 2.

TRPV1+ neuron activation confers resistance to S. mansoni and induces cytokine expression in γδ T cells. (A) Experimental approach for optogenetic ear stimulation of ChR2 control or TRPV1-ChR2 mice followed by evaluation of skin resistance after exposure to 150–200 S. mansoni cercariae for 30 min or lung larval (schistosomulae) load 6 d post-infection with 500 S. mansoni cercariae. Life cycle is depicted for reference. (B) Ear thickness measured daily during photostimulation of ChR2 control or TRPV1-ChR2 mice. (C) Percentage of non-penetrating cercariae from ChR2 control or TRPV1-ChR2 mice that were previously photostimulated as shown in panel A. (D) Lung schistosomulae of light-stimulated ChR2 control or TRPV1-ChR2 mice quantified 6 d post-infection. (E) Percentage of IL-17+ cells in the skin of control or TRPV1-ChR2 mice after 5 d of blue light stimulation followed by S. mansoni infection. (F), Pie charts depicting the percentage of lymphocyte cellular subsets within IL-17+ cells in the skin of mice from E. (G, H), Representative dot plots and quantification of IL-17+ dermal γδ T cells (CD45+ CD90+ TCRδ+) and (I, J), CD4+ Th cells (CD45+ CD90+ TCRβ+CD4+Foxp3-) by flow cytometry of control or TRPV1-ChR2 mice optogenetically stimulated for 5 d followed by S. mansoni infection for 1 d. (K, L) Representative dot plots, percentages, and absolute numbers of Ki-67+ and (M, N) IL-13+ γδ T cells quantified by flow cytometry. P values were determined by 2-tailed Student t-tests, or 2-way ANOVA with post hoc correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative of 2-3 independent experiments, each with ≥4 biological replicates. Panel A was created in Biorender.

Figure 3.

TRPV1+ neurons promote neutrophil and monocyte recruitment and activation after S. mansoni infection. (A) Ear thickness of light-stimulated control or TRPV1-ChR2 mice measured 1 d after exposure to S. mansoni cercariae. (B), Representative dot plots of neutrophils (CD11b+ Ly6G+ Ly6Cint) and monocytes (CD11b+ Ly6G- Ly6Chi) assessed by flow cytometry of light-stimulated control or TRPV1-ChR2 mice 1 d after S. mansoni infection. (C, D) Percentage and absolute numbers of neutrophils and monocytes from S. mansoni-infected control or TRPV1-ChR2 mice. (E–G) Representative dot plots and quantification of iNOS expression in neutrophils and monocytes assessed by flow cytometry of light-stimulated control or TRPV1-ChR2 mice 1 d after S. mansoni infection. (H) Representative histogram and gMFI quantification of iNOS expression in neutrophils and (I), monocytes in the skin of light-stimulated control or TRPV1-ChR2 mice 1 d after S. mansoni infection. P values were determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative of 2–3 independent experiments, each with ≥4 biological replicates.

Figure 4.

TRPV1+ neurons are required to mitigate S. mansoni lung larval burden, γδ T cell responses and myeloid cell skin accumulation. (A) Thermal pain sensitivity in the paws of wildtype mice treated with vehicle or resiniferatoxin (RTX) assessed by withdrawal times (seconds) to infrared heat. (B, C) Percentage of non-penetrating cercariae and d6 lung larval burden from vehicle or RTX-treated mice infected with S. mansoni. (D) Percentage and absolute numbers of IL-17+, (E) Ki67+, and (F), IL-13+ γδ T cells 1 day post-infection with S. mansoni in vehicle or RTX-treated mice. (G, H) Skin neutrophils and monocytes from vehicle- or RTX-treated mice infected with S. mansoni. P values were determined by two-tailed Student’s t-test. *P < 0.0 5, **P < 0.01, ***P < 0.001, ****P < 0.0001. Representative of 2–3 independent experiments, each with ≥5 biological replicates.