Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity

Abstract

Cutaneous TRPV1⁺ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1⁺ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1⁺ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1⁺ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1⁺ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Keywords: C. albicans; CGRP; S. aureus; TRPV1; antidromic; host defense; innate immunity; neuroimmunology; optogenetics; skin.

Fig. 1. Optogenetic activation of TRPV1⁺ sensory neurons induces cutaneous inflammation.

(A) Immunofluorescent visualization of TRPV1-Ai32 flank skin illustrates dermal and epidermal expression of ChR2/eYFP (green), PGP9.5+ nerve fibers (red), and DAPI nuclear label (blue). Scale bar, 30 μm. (B) Summary of the percent of lumbar and thoracic DRG cell bodies from TRPV1-Ai32 mice identified as peptidergic (TRPV1, GFRα3) and nonpeptidergic (IB4, GFRα2) that co-express ChR2-eYFP. (C) Representative images of ears from Ai32 and TRPV1-Ai32 mice after 5 days of photostimulation (+λ). (D) Ear thickness and (E) inflammation score measured at the indicated time points during photostimulation of Ai32 (black line) and TRPV1-Ai32 (red line) mice. (F) Ear thickness and (G) inflammation score measured at the indicated time points during photostimulation of RTX (blue line) or control-treated (red line) TRPV1-Ai32 mice. Each symbol in (B) represents counts from a single DRG section from an individual animal. For D-G, results are represented as mean with a group size of 5-7 mice. Results in all panels are represented as mean ± SEM from 2-4 independent experiments. Significance was calculated using unpaired Student’s t-test, *p<0.05, **p<.001, ***p<.0001. See also Figure S1 and S2.

Fig. 2. TRPV1⁺ neuron activation is sufficient to initiate Type-17 inflammation.

(A) Representative histological sections of ears from Ai32 and TRPV1-Ai32 mice after 5 days of photostimulation (+λ). Note acanthosis, parakeratosis and dense neutrophilic and lymphocytic infiltrate in photostimulated TRPV1-Ai32 mice. Scale bars, 200 μm. (B) Total numbers of the indicated cell type assessed by flow cytometry from Ai32, TRPV1-Ai32 and RTX treated TRPV1-Ai32 mice after 4 days of photostimulation. (C) Il23a, Il6, Tnfα mRNA levels in whole photostimulated (Ipsilateral, dark bars) or sham treated (contralateral, white bars) ears from Ai32 (open circles) or TRPV1-Ai32 (red squares) mice isolated at 6 hours after initiation of photostimulation. (D) IL-23, IL-6, and TNFα protein levels in whole photostimulated ears from Ai32 or TRPV1-Ai32 mice isolated at 48 hours after initiation of photostimulation. (E) As in C except that ears were harvested at the indicated time point following initiation of photostimulation. Each symbol in B-D represents data from an individual animal. Each symbol in E represents mean in a group size of 5-8 animals. Results in all panels are represented as mean ± SEM from a minimum of 3-4 individual experiments. Significance was calculated using a one-way ANOVA (B, C, E) or an unpaired Student’s t-test (D) , *p<0.05, **p<.001, ***p<.0001. See also Figure S3.

Fig. 3. TRPV1⁺ neuron activation induces expansion of IL-17-producingTCRγδ and CD4 T cells

(A) Representative flow cytometry plots and (B) summary data of IL-17A expression in PMA/Ionomycin stimulated ILC, TCRγδ and CD4 T cells isolated from Ai32 (black circles) and TRPV1-Ai32 (red squares) ears following 4 days of photostimulation. (C) Quantification of Evans Blue dye extravasation 30 minutes following ear photostimulation of Ai32 control (open circles) and TRPV1-Ai32 (red squares) mice. (D) As in fig. C except TRPV1-Ai32 mice were treated i.p. with anti-IL17A mAb (blue squares) or Isotype control (red squares) prior to photostimulation. E) Ear thickness and (F) inflammation score measured at the indicated time points during photostimulation of anti-IL17A mAb (blue squares) or isotype control (red squares) treated TRPV1-Ai32 mice. (G) Representative flow cytometry plots and (H) summary data of total numbers of the indicated cell type assessed by flow cytometry from anti-IL17A mAb (blue squares) or Isotype control (red squares) treated TRPV1-Ai32 mice. Each symbol in B, C, D, and H represents data from an individual animal. Each symbol in E and F represents mean in a group size of 5-8 animals. Results in all panels are represented as mean ± SEM from 2-3 individual experiments. Significance was calculated using unpaired Student’s t-test *p<0.05, **p<.001, ***p<.0001.

Figure 4:. Vesicle-mediated release of CGRP is required for TRPV1⁺ neuron-induced Type-17 inflammation.

(A) Il23a, Il6, Tnfα mRNA expression in whole photostimulated (“+”) or sham treated (“−”) ears from TRPV1-Ai32 mice pre-treated with vehicle (red squares) or 0.3U Botulinum toxin (BoNT/A) (blue squares) isolated at 6 hours after initiation of photostimulation. (B) ELISA quantification of CGRPα in supernatants from cultured DRG isolated from Ai32 (black circles) or TRPV1-Ai32 (red squares) mice after 30 minutes of sham (−) or photostimulation(+). (C) ELISA quantification of CGRPα in supernatants from ex vivo skin explant organ cultures of Ai32 or TRPV1-Ai32 mice harvested 6 hours after initiation of in vivo photostimulation. (D) Il23a, Il6, Tnfα mRNA expression in whole photostimulated (“+”) or sham treated (“−”) ears from TRPV1-Ai32 mice pre-treated with vehicle (red squares) or 0.5ug CGRP_8-37 (blue squares) isolated at 6 hours after initiation of photostimulation. (E) As in D except that ears were harvested at the indicated time point following initiation of photostimulation. (F) Summary data of IL-17A expression by PMA/Ionomycin stimulated TCRγδ and CD4 T cells isolated from ears of TRPV1-Ai32 and Ai32 mice treated daily with vehicle or CGRP_8-37 during 4 days of photostimulation. Each symbol in A, C, D, and F represents an individual animal. Each symbol in B represents pooled DRG neurons from an individual mouse. Each symbol in E represents the mean in a group size of 4-7 mice. Results in all panels are represented as mean ± SEM from 2-3 independent experiments. Significance was calculated using a one way ANOVA, *p<0.05, **p<.001, ***p<.0001.

Fig. 5. TRPV1⁺ neuron activation generates a nerve reflex arc.

(A) Scheme depicting location of photostimulation (λ), injection, and visualization. (B) Representative 2D laser doppler image taken before (−λ) and after (+λ) 30 minutes of photostimulation in Ai32 and TRPV1-Ai32 mice injected with vehicle or 5mM bupivacaine at the Stim site. Stim site indicated by solid circle; Adj site indicated by dashed circle. The rate of blood flow is depicted from low flow (blue) to high flow (red). (Scale bars, 4mm) (C) Summary of perfusion units in the indicated skin regions as depicted in B. Each symbol represents data from individual animals. All data is represented as mean ± SEM from 3 independent experiments. Significance was calculated using a Student’s t-test, **p<.001. See also Figure S4.

Fig. 6. TRPV1⁺ nerve activation provides regional Type-17 inflammation and host defense via a nerve-reflex arc

A) Il23a, Il6, Tnfα mRNA levels in flank skin isolated from photostimulated Ai32 (open circles) or TRPV1-Ai32 (red squares) mice at the indicated skin site 6 hours after initiation of photostimulation. (B) C. albicans and (C) S. aureus CFU is shown from the indicated skin region of epicutaneously infected Ai32 or TRPV1-Ai32 mice on day 3 post-infection with photostimulation on days 0, 1, and 2. (D) CGRP protein and (E) Il23a, Il6, Tnfα mRNA levels at the indicated site from photostimulated TRPV1-Ai32 mice pre-treated with 5mM bupivacaine (blue squares) or vehicle (red squares) at the Stim site. (F-G) As in (D-E) except TRPV1-Ai32 mice were pretreated at the stim site with 0.3U Botulinum toxin (BoNT/A) or vehicle. (H) C. albicans CFU is shown from the indicated skin region of TRPV1-Ai32 mice treated twice daily with vehicle (red squares) or 5mM Bupivacaine (blue squares) at the Stim site. Tissue harvested on day 3 post-infection with photostimulation on days 0, 1, and 2. (I) As in (H) except that 0.5ug CGRP_8-37 (blue squares) or vehicle (red squares) was injected daily at the adjacent site. (J) As in (H) except mice were pretreated i.p. with neutralizing mAbs to IL-17A (blue squares) or isotype control (red squares). Each symbol represents an individual animal. Results in all panels are represented as mean ± SEM from 2-4 independent experiments. Significance was calculated using a Student’s t-test. *p<0.05, **p<.001, ***p<.0001. See also supplemental figure S5

Fig. 7. TRPV1⁺ neurons provide anticipatory immunity to C. albicans

(A) Heat killed C. albicans (HKCA) mixed with 5mM bupivacaine (blue symbols) or vehicle (red symbols) was administered i.d. to WT C57BL/6 mice (injected site labeled HKCA). Il23a, Il6, Tnfα mRNA levels in flank skin at the indicated site 6 hours after injection is shown. (B) Scheme depicting intradermal HKCA treatment of mice then epicutaneously infected with GFP-expressing C. albicans. 10⁶ HKCA in 10 ul mixed with either vehicle or 5mM bupivacaine was administered i.d. 2 hrs before epicutaneous infection of WT mice with C. albicans-GFP. Mice were treated i.d. with 5mM bupivacaine or vehicle at the HKCA-injected site twice daily. The injection site is indicated by the solid white circle. (C) Representative in vivo fluorescent images of GFP fluorescence (representing active C. albicans infection) on days 1, 2, and 3 of infection taken using an IVIS in vivo imaging system. Scale bar 4mm. Intensity of GFP ranges from dull red (low) to bright yellow (high). (D) Summary data of the area of reduced GFP fluorescence (defined as 50% of the maximum fluorescence) as depicted in panel C. Each symbol in A represents data from individual animals. Each symbol in D represents mean in a group size of 5-8 animals. All data are represented as mean ± SEM from three experiments. Significance was calculated using a one way Student’s t-test, *p<0.05, **p<.001, ***p<.0001. See also Figure S6.