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House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation

Allergic skin diseases, such as atopic dermatitis (AD), are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely-distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine-protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons, that expressed the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDMs-activated nociceptors drove the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors through the release of substance P and the activation of the cationic molecules receptor MRGPRB2 on mast cells. This data indicates that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor-MRGPRB2+ sensory clusters represents a key early event in the development of allergic skin reaction.

appropriate behavioral responses such as withdrawal (to avoid tissue injury) and scratching (to remove irritants). Nociceptors may be involved in manifestations of AD by promoting itch and scratching behavior 13,15 , an idea supported by clinical studies showing that the serum of patients with AD exhibits high amounts of neuropeptides, such as the cationic neuropeptide substance P (SP), and that the amounts detected correlate with the severity of the disease 15,16 . While much remains to be learned about itching sensations, peripheral neurons also have the potential to influence immune cells and inflammatory responses [17][18][19][20][21][22] .
Mast cells are innate immune cells thought to be involved in allergic diseases, including AD, in which they recognize a specific antigen through their high-affinity receptors for IgE (FcεRI) 23 . Under homeostatic conditions, skin and peritoneal mast cells in mice specifically express MRGPRB2, a receptor for cationic molecules from the Mas-related G proteincoupled receptors family 24 . MRGPRX2, the human ortholog of MRGPRB2, and not the neurokinin 1 receptor, is the main receptor for cationic molecules, including SP, in human skin mast cells 25,26 . MRGPRX2 or MRGPRB2-mediated activation of mast cells can result in a remarkably fast degranulation dynamics, which is associated with the development of rapid and localized mast cell-dependent inflammation 25 .
Whether specialized neuro-immune cross-talk has a role in the development of allergic skin inflammation remains unclear. Combining genetically-modified mouse models with intravital imaging approaches, we show here that a large proportion of TRPV1 + nociceptor projections form physical contacts with MRGPRB2 + mast cells. These neuro-immune clusters can sense the presence of allergic cues and can trigger pathogenic type 2 immune responses and associated lesions in the skin.

Tac1 expression is required for allergic skin inflammation
We used a mouse model of allergic skin inflammation driven by six epicutaneous exposures over one month to two antigens found together in the lesional skin of 80-100% of AD patients [2][3][4][5][6][7]26 , 10 μg of the HDM strain Dermatophagoides farinae (D. farinae) and 500 ng of the bacterial exotoxin SEB from S. aureus ( Supplementary Fig. 1a,b). Compared to healthy subjects, AD patients often suffer from S. aureus skin infections which are thought to play an important role in the pathogenesis and/or worsening of AD 6 . This model recapitulates moderate to severe AD-like disease, associated with both the histopathological features of an exacerbated type 2 immune response and a global gene expression pattern statistically similar to that seen in human AD 8,27 . Wild-type mice sensitized with a combination of SEB and D. farinae developed more severe skin inflammation than those treated with SEB or D. farinae alone, while D. farinae alone induced more skin lesions than SEB alone ( Supplementary Fig. 1c,d). Compared to vehicle, treatment with D. farinae + SEB induced a systemic D. farinae-specific T H 2 response ( Supplementary Fig. 1e,f). In accord with reports that patients with moderate to severe AD were treated successfully with dupilumab 28 , a human monoclonal antibody against the IL-4 receptor α (IL-4Rα) that inhibits both IL-4 and IL-13 signaling, systemic treatment with blocking antibodies for the mouse IL-4Rα significantly decreased the skin lesions and the histopathological features in the AD-like mouse model ( Supplementary Fig. 1g,h). These data indicate that treatment with D. farinae + SEB leads to the development of AD-like allergic skin inflammation dependent on type 2 cytokines.
The neuropeptide SP is thought to be released primarily from a unique subpopulation of TRPV1 + TRPA1peptidergic nociceptors that highly express Tac1 29 , the gene encoding the SP precursor. Using publicly available gene expression data 30 , we mapped the expression of Trpv1, Tac1 and Trpa1 among different mouse tissues and various subpopulations of immune cells. Trpv1, Tac1 and Trpa1 were highly (or exclusively, in the case of Trpv1 and Trpa1) expressed in dorsal root ganglia (DRG), where the somas of sensory neurons are located, with weak additional expression of Tac1 in the central and the enteric nervous systems ( Fig. 1a and Supplementary Fig. 1i). In whole-mounted skin biopsies from C57BL/6J wild-type mice, SP expression was restricted to PGP9.5 + cutaneous neuronal fibers (Fig. 1b).
To analyze the role of SP in the development of allergic skin inflammation, we treated wildtype and Tac1 -/mice with D. farinae + SEB and assessed the development of key pathological features associated with AD 8 . Compared to vehicle-treated wild-type mice, D. farinae + SEB-treated wild-type mice developed macroscopic skin lesions (Fig. 1d), increased epidermal thickness, strong infiltration of eosinophils and neutrophils (Fig. 1e) and elevated serum D. farinae-specific IgG 1 and IgE (Fig. 1f), along with a profound alteration of filaggrin protein expression (Fig. 1g,h), a key component of skin barrier function linked to human AD 12 . In addition, D. farinae + SEB-treated wild-type mice had increased expression of keratin 6 (K6), a marker of inflammatory stress in keratinocytes ( Supplementary Fig. 2a,b) and alterations in claudin-1, K14 and K10 expression (Supplementary Fig. 2c-h). Expressions of two other structural proteins loricrin and Ecadherin were not significantly affected ( Supplementary Fig. 2i-l). By contrast, D. farinae + SEB-treated Tac1 -/mice were mostly protected from disease, with substantial reduction in skin lesion development, histological abnormalities, infiltration of immune cells, serum levels of D. farinae-specific IgG 1 and IgE and changes in skin barrier architecture (Fig. 1c-h and Supplementary Fig. 2). Taken together, this data suggested that expression of Tac1 and SP was restricted to the neuronal compartment of the skin and that expression of Tac1 was required for the full development of the pathological features associated with allergic skin inflammation in this model.

HDMs directly activate substance P-producing TRPV1 + neurons
To analyze the role of TRPV1 + nociceptors in the development of allergic skin inflammation, we treated wild-type mice systemically with resiniferatoxin (RTX), which is a potent TRPV1 agonist selectively ablating TRPV1 + nociceptors 17,18 (Supplementary Fig.  3a,b). RTX-treated mice and control DMSO-treated mice were subsequently treated with D. farinae + SEB to induce allergic skin inflammation. RTX-treated mice had a strong reduction of skin lesions (Fig. 2a,b) and lesion-associated histopathological features compared to control DMSO-treated mice (Fig. 2c), along with restored filaggrin organization and decreased expression of the stress marker K6 ( Supplementary Fig. 3), suggesting that TRPV1 + nociceptors were required for the full development of allergic skin inflammation in this model. The primary function of nociceptors is to detect potentially damaging stimuli and initiate appropriate behavioral responses such as removal or scratching. We investigated whether D. farinae and SEB were directly detected by TRPV1 + DRG neurons cultured ex vivo. We found that 5 ng/ml SEB alone induced only a weak (barely detectable in some experiments) influx of calcium in DRG neurons (Fig. 2d,e), while concentrations ranging from 5 ng/ml of D. farinae, either alone (Fig. 2d,f) or in combination with 5 ng/ml SEB (Fig. 2d,g) triggered an increase in intracellular calcium in a subpopulation of DRG neurons that also responded to the TRPV1 agonist capsaicin (Fig. 2h,i).
D. farinae alone or in combination with SEB also triggered the secretion of SP from DRG neurons, with a tendency, in some experiments, for D. farinae + SEB to trigger less secretion of SP compared to either alone (Fig. 2j). Moreover, cultured DRG neurons from RTXtreated mice did not secrete SP in response to D. farinae alone or D. farinae + SEB, while DRG neurons from DMSO-treated mice secreted SP (Fig. 2k). Importantly, DRG neurons from RTX-treated mice were also unresponsive to capsaicin (Fig. 2k) indicating that in vivo RTX-treatment efficiently depleted TRPV1 + neurons. These data indicated that D. farinae directly triggered the secretion of SP from TRPV1 + DRG neurons cultured ex vivo.
Next, we investigated by which mechanism D. farinae activated TRPV1 + DRG neurons.
Treatment of DRG neurons with NBP2-29328, a MYD88 inhibitory peptide 8 , did not prevent D. farinae-induced activation of DRG neurons compared to DRG neurons treated with a non-relevant control peptide ( Supplementary Fig. 4a,b), indicating that, at least under the culture conditions used here, DRG neurons activation was not dependent on MYD88dependent signaling through Toll like receptors (TLR). Next, we investigated whether the proteolytic activity of D. farinae was required for triggering DRG neuron activation ex vivo. Like other common environmental allergens, HDM strains, including D. farinae 31 have intrinsic proteolytic activity that can degrade epithelial junctions [32][33][34][35] or full-length IL-33 36 in vivo. To characterize the type of proteolytic activity present in the D. farinae extracts used, we measured the proteolytic activities of D. farinae with or without specific and irreversible inhibitors of cysteine or serine protease activities. Pretreatment with the cysteine protease inhibitor E64, but not with the serine protease inhibitor AEBSF, completely inhibited the proteolytic activity of D. farinae, at levels comparable to heat-inactivation of D. farinae (Fig. 3a). No proteolytic activity was measured in the absence of dithiothreitol (DTT), a reducing agent required for cysteine protease activity in cell-free conditions (Fig.  3a), indicating the presence of cysteine protease activities in the D. farinae extract. SEB alone neither exhibited protease-like activity nor enhanced D. farinae proteolytic activity ( Supplementary Fig. 4c).
Heat-induced inactivation or treatment with E64 prevented the influx of calcium in TRPV1 + DRG neurons by D. farinae ex vivo ( Fig. 3b-e). However, TRPV1 + DRG neurons activation was independent of the expression of the protease-activated receptor PAR2 by the neurons (Supplementary Fig. 4a,b). We next investigated whether other clinically-relevant environmental allergens with either predominant cysteine protease activity, such as the HDM strain D. pteronyssinus 31 or serine-like protease activities 36 from pollen (ragweed), fungus (Alternaria alternata) or the German cockroach could also directly activate DRG neurons. Only D. pteronyssinus triggered significant influx of calcium in DRG neurons, comparable to those obtained after stimulation with D. farinae (Fig. 3f). These data indicate that two common HDM strains with predominant cysteine protease activity involved in allergic inflammation and suspected to have major roles in various allergic disorders, could trigger the activation of a subset of nociceptors directly, predominantly via a cysteine protease activity-dependent mechanism.

Functional MRGPRB2 is required for allergic skin inflammation
Mouse MRGPRB2 and its human ortholog, MRGPRX2 are mast cell-restricted receptors for several cationic substances, including the neuropeptide SP. We used Kit-dependent Kit W-sh/W-sh mast cell-deficient mice, in which KIT, the receptor for the main mast cell growth and survival factor SCF is mutated, and Kit-independent Cpa3-Cre To assess whether MRGPRB2 contributed to the development of allergic skin inflammation, we used Mrgprb2 mut/mut mice, in which MRGPRB2 is genetically inactivated by mutation, but have normal numbers of mast cells 24 ( Supplementary Fig. 6g,h). In contrast to Mrgprb2 +/+ littermates, Mrgprb2 mut/mut mice did not develop allergic skin inflammation after treatment with D. farinae + SEB, and had substantial reductions in skin lesions (Fig. 4a,b), histological abnormalities, infiltration of eosinophils and neutrophils (Fig. 4c)

TRPV1 + nociceptors activate skin mast cells through MRGPRB2
We next explored whether D. farinae and SEB triggered the degranulation of dermal mast cells in vivo through the activation of TRPV1 + Tac1 + nociceptors and MRGPRB2. A single intradermal (i.d.) injection of sulforhodamine 101-labeled avidin (Av-SRho) can be used to monitor skin mast cell granule structures specifically in live mice by two-photon microscopy or ex vivo by confocal microscopy 37 . We injected vehicle or 1 μg D. farinae + 50 ng SEB i.d. into the ear pinna of Av.SRho-injected mice and measured tissue swelling, as an early readout of inflammation and Av.SRho + mast cell degranulation in whole-mounted ear skin using ex vivo volumetric confocal microscopy 45 minutes post D. farinae + SEB injection ( Supplementary Fig. 8a). I.d. injection of vehicle induced very mild swelling of the ear tissue ( Fig. 5a) and did not trigger substantial skin mast cell degranulation (Fig. 5b,c) in DMSO-treated wild-type mice or Mrgprb2 +/+ mice, while injection of D. farinae + SEB induced strong ear swelling (Fig. 5a) and abundant degranulation of Av.SRho + mast cells (Fig. 5b,d). After injection of D. farinae + SEB, RTX-treated wild-type mice or Mrgprb2 mut/mut mice had a substantial reduction of both ear swelling (Fig. 5a) and degranulation of Av.SRho + mast cells (Fig. 5b,d) compared to DMSO-treated wild-type or Mrgprb2 +/+ mice. Wild-type mice injected i.d. with blocking antibodies against SP had a strong reduction of both ear swelling (Fig. 5e) and mast cell degranulation ( Fig. 5f,g) compared to IgG isotype-treated control mice. In addition, a 30 minute pretreatment of D. farinae + SEB with the cysteine protease inhibitor E64 decreased ear swelling significantly ( Supplementary Fig. 8b,c). D. farinae and/or SEB did not trigger the degranulation of in vitro-cultured mouse or human mast cells at the tested concentrations ( Supplementary Fig.   8d,e).
We next tested whether activation of TRPV1 + nociceptors in the skin using the TRPV1 agonist capsaicin would result in MRGPRB2-dependent mast cell degranulation in vivo. I.d.
injection of 1 μM capsaicin did not induce significant ear tissue swelling (Fig. 5h) or Av.SRho + mast cell degranulation (Fig. 5i,j) in RTX-treated wild-type mice or Mrgprb2 mut/mut mice, while strong ear swelling (Fig. 5h) and linked degranulation of Av.SRho + mast cells (Fig. 5i,j) were observed in DMSO-treated wild-type and Mrgprb2 +/+ mice, indicating that the effect was largely dependent on TRPV1 + nociceptors and MRGPRB2. These results indicate that, upon i.d. injection of D. farinae + SEB or capsaicin, the observed mast cell degranulation and associated early signs of inflammation require functional TRPV1 + nociceptors, release of SP and signaling through MRGPRB2.
Next, we adapted the fluorescent avidin-based mast cell imaging approach to visualize the activation of TRPV1 + nociceptors and the activation of mast cells simultaneously using intravital two-photon microscopy 37 . For this purpose we used Pirt-GCaMP3 mice, in which expression of the calcium tracer GCaMP3 is driven by the Pirt promoter in sensory neurons 38  ng SEB, either in combination or separately, in Av.SRho-labelled Pirt-GCaMP3 mice we observed a significant increase in GCaMP3 fluorescence in skin neurons compared to vehicle-injected mice (Fig. 6a,b) and degranulation of ~ 50% Av.SRho + mast cells (Fig.  6a,c), suggesting that the presence of D. farinae and/or SEB antigens in the dermis could lead to the activation of TRPV1 + nociceptors and mast cells in vivo. Finally, we used an automated computer-assisted calculation method to perform an unbiased analysis of the spatial organization of activated TRPV1 + nociceptors and Av.SRho + mast cells in the dermis of Pirt-GCaMP3 mice as assessed by live imaging. About 62 % of Av.SRho + mast cells either formed contacts with activated TRPV1 + nociceptors in the skin (25%) or resided in close proximity to activated nociceptors (37% of Av.SRho + mast cells were within less than 25 μm from activated TRPV1 + nociceptors) (Fig. 6d-f). These data suggest that mast cells and TRPV1 + nociceptors formed cellular clusters in the skin that can be activated in the

Discussion
Here we found that TRPV1 + Tac1 + nociceptors and MRGPRB2 + mast cells formed clusters in the mouse skin that could be activated by allergens, such as HDMs. This, in turn, drove the development of type 2 immunity-associated skin inflammation in mice that mimicked many features of human AD. In addition, our results identified MRGPRB2 as a key receptor that facilitated communication between skin mast cells and TRPV1 + Tac1 + nociceptors.
Based on the generated high-resolution 3D images, it is interesting to speculate that the close anatomical co-localization of mast cells and TRPV1 + nociceptors in the mouse dermis might enable the accumulation of high enough amounts of secreted neuropeptide such as SP to reach the previously reported high activation threshold of MRGPRB2 39 . Fixed biopsies have indicated proximity or contact between nerve fibers and mast cells in mice, pigs and humans [40][41][42][43] , while mouse and human mast cells can be directly activated by neuropeptides (including SP) in vitro 25,42,44 . A bi-directional activation process between mast cells and submucosal neurons in cultured gut biopsies has been reported 45 . Our study offers direct evidence of functional interactions between nociceptors and mast cells in vivo and identified these interactions as a critical element in the development of type 2 skin inflammation. Targeting SP-enabled interactions between TRPV1 + nociceptors and MRGPRX2 + mast cells might therefore represent a promising therapeutic approach for the prevention and/or treatment of human AD pathology.
Recent studies have reported that MRGPRB2 on mast cells has an important role in the regulation of inflammatory pain 46 and itch sensation 47 . SP and MRGPRB2 were reported to be directly involved in the regulation of inflammatory mechanical and thermal hyperalgesia, and in the recruitment of innate immune cells at the site of injury 46 . In addition, compared to activation through canonical FcεRI, mast cell activation through MRGPRB2 induces itch distinct from classical histaminergic itch 47 . MRGPRB2-mediated activation of mast cells results in the differential release of granule-associated mediators (including some with known pruritogen function such as histamine, serotonin or tryptase), and MRGPRB2 deficiency significantly decreases itch in different models of allergic contact dermatitis, which is thought to be associated with pathogenic CD8 + T cell responses 48 . These observations indicate that MRGPRB2 activation on mast cells modulates the transmission of sensations of pain and itch in different inflammatory contexts.
Mast cells can drive certain types of IgE-independent pseudo-allergic reactions through the binding of a wide range of cationic drugs (such as the FDA-approved mivacurium or icatibant) to MRGPRX2 or MRGPRB2 24,49,50 . It is tempting to speculate that MRGPRX2 or MRGPRB2-mediated mast cell activation by cationic drugs could lead to the activation of nociceptors at the site of injection and the subsequent development of pain and/or itch. However, more in vivo work is needed to define whether the clusters of TRPV1 + Tac1 + Europe PMC Funders Author Manuscripts nociceptors and MRGPRB2 + mast cells described here are involved in drug-induced adverse events.
We found that D. farinae, but not SEB, directly activated TRPV1 + DRG neurons ex vivo through a cysteine protease-dependent mechanism. We can't rule out the possibility that direct effects of D. farinae and/or SEB on skin mast cells, apart from effects on degranulation, also may have contributed to our findings. However, the data presented suggest that, under the conditions studied, D. farinae + SEB-induced cutaneous inflammation required TRPV1 + nociceptors and the release of SP, which then activated skin mast cells.
Most patients with severe AD are colonized with exotoxin-producing S. aureus 4,6,7 . In agreement to that, we found that D. farinae in combination with SEB triggered the development of more severe skin inflammation than when the two antigens were used separately. While SEB alone did not activate DRG neurons cultured ex vivo, after i.d. injection in vivo it triggered GCaMP3 fluorescence in skin neurons comparable to that observed after injection of D. farinae. This might indicate that SEB activated nociceptors through an indirect mechanism; however, the precise mechanism(s) remain(s) to be elucidated. It is possible that SEB, which is known to be a potent superantigen, could also favor the proliferation of T cells in our model. However, SEB treatment alone in wild-type mice triggered less severe skin inflammation and skin lesions than treatment with D. farinae alone. It is therefore possible that a direct mechanism of nociceptor activation by D. farinae cysteine protease activity might be required to efficiently trigger the development of this model of D. farinae + SEB-induced allergic skin inflammation.
The sensing of allergen-associated proteolytic activity has been proposed to be an important mechanism of environmental allergen detection that could contribute to the initiation of allergic diseases 31,36 . Our data suggest that the clusters of TRPV1 + nociceptors and MRGPRB2 + mast cells might represent tissue-resident "sensory systems" that would detect allergens with cysteine-like protease activity in the skin and initiate type 2 immunityassociated allergic skin disease. Sensory nerve fibers and mast cells are also present in the upper airways, lungs and gastro-intestinal tract, which are organs continuously exposed to environmental allergens and within which allergic diseases are also known to develop. However, the extent to which neuron-mast cell clusters are involved in the development of other allergic disorders in different organs remains to be investigated.

Mice
Four-to 8-week-old C57BL/6J and Tac1 -/mice were purchased from Charles River or the

Model of allergic skin inflammation
Allergic skin inflammation was induced as previously described 8,27 (described in Supplementary Fig. 1). Briefly, back skin was shaved and a solution of 500 ng of Staphylococcal enterotoxin B (SEB, Sigma-Aldrich) and of 10 μg of Dermatophagoides farinae extract (HDM, Stallergen Greer) in PBS was applied on a gauze pad placed on the shaved back and occluded with a Tegaderm™ Transparent Dressing (3M HealthCare). Three days later, the gauze pads were replaced. Mice were monitored on a daily basis and if a mouse removed the bandage, a new dressing was immediately applied on this mouse and all of the other mice within the same experiment (so that they received the same treatment and equal amounts of antigens). Four days later, dressings were removed and mice were kept without treatment for the next week. This "3 + 4 days" pattern of treatment was repeated two more times, so that the mice were subjected to three cycles of such treatment. Two days after the last cycle of treatment, the mice were euthanized and back skin specimens corresponding to the treated areas were obtained for analyses. In a set of experiments, mice were also treated with either IgG isotype control or anti-CD124 antibody (100 μL; i.p.) at 2.8 mg/kg twice a week for the entire period of treatment. The last treatment with either of these antibodies was done one day before sacrifice. This model has been efficiently used in both Inserm Toulouse and Stanford University and key experiments have been repeated in both animal facilities.

Ablation of Trpv1 + nociceptors in mice
To ablate Trpv1 + nociceptors, 4-week-old C57BL/6J mice were subcutaneously injected with increasing resiniferatoxin (RTX) doses of 30 μg/kg, 70 μg/kg and 100 μg/kg in 100 μL of PBS for 3 consecutive days. Control mice were injected with similar volumes of DMSO in 100 μL of PBS. 4 weeks later, denervation was assessed using the classical tail flick assay.

Bone marrow-derived cultured mouse mast cells (BMCMCs)
Hematopoietic cells were collected from C57BL/6 mouse femurs and cultured in DMEM supplemented with L-Glutamax, 2-β-mercaptoethanol (50 μM; Invitrogen), murine IL-3 (10 ng/mL; Peprotech), 10% Fetal Bovine Serum (FBS, Invitrogen) and Peni/Strep (100X). Twenty-four hours later, adherent cells were removed, and fresh culture medium was added to non-adherent cells. Three days later, cells were resuspended in fresh culture medium. The same procedure was repeated twice a week. Cells were used for experiments between week 4 and week 10. Nat Immunol. Author manuscript; available in PMC 2020 April 07.
Europe PMC Funders Author Manuscripts concentrations of D. farinae and SEB (5 ng/mL, 50 ng/mL and 500 ng/mL), alone or together. β-Hexosaminidase release in the supernatants was measured as previously described 25 .

Intracellular flow cytometry of D. farinae-specific CD4 + T cells
Following induction of allergic skin inflammation, spleens from vehicle-treated or D. farinae + SEB-treated mice were harvested and dissociated to obtain a suspension of cells. 200,000 spleen cells were stained with CFSE for 7 minutes at 37°C and incubated for 5 days with 10 μg/ml of D. farinae in RPMI 1640 supplemented with 10% FCS GlutaMAX-I, sodium pyruvate, 2-mercaptoethanol, and 10μg/ml ciprofloxacin. Intracellular cytokines were analyzed by gating on proliferating (CFSE low ) CD4 + T cells after a 5-hour restimulation with phorbol 12-myristate 13-acetate (50 ng/mL, Sigma) and ionomycin (1 mg/mL, Sigma) in the presence of GolgiStop (BD Pharmingen). Cells were fixed, permeabilized (0.1% saponin in PBS 0.5% BSA), and stained with antibodies directed against mouse IL-4, IL-5, IL-13 or IFN-γ. Flow cytometric data were acquired on a BD FACSCanto cytometer and were analyzed using FlowJo software (Tree Star, Inc, Ashland, Ore).

Skin section preparation, histology, immunofluorescence, and confocal microscopy
Mouse back skin (1-2 cm 2 ) samples were fixed in 10% formalin and embedded in paraffin. Four-micrometer-thick sections were stained with H&E, and photographs were taken using a Nikon H600L microscope and analyzed with NIS-Elements imaging Software. All sections were "coded" so the evaluator was not aware of their identity, as previously described 8 . Eosinophils and neutrophils were counted on H&E-stained skin sections from all mouse strains. For immunostaining of mouse specimens, 4-μm-thick sections were pretreated using a heat-induced epitope retrieval method 8 in 10 mM sodium citrate buffer (pH 6.0), then permeabilized for 30 minutes in PBS supplemented with 0.5% BSA and 0.1% saponin. Permeabilized skin sections were incubated overnight at 4°C with primary antibodies, extensively washed, and incubated with appropriate secondary antibodies for 2 hours at room temperature in the dark. Images of 1024 x 1024 pixels were acquired using a Zeiss LSM780 and LSM710 Meta inverted confocal laser-scanning microscopes. Images were processed using Zen software (Zeiss). Epidermal K6, K17, Claudin 1, Filaggrin, Loricrin and E-cadherin mean fluorescence intensities were analyzed using the "measurement function" of ImageJ software on randomly chosen epidermal areas of identical size (i.e., same total number of pixels).

Amounts of serum D. farinae-specific IgG1 and IgE antibodies by ELISA
Each incubation step described below was followed by 3 to 5 washing steps using PBS containing 0.05% Tween-20. MaxiSorp ELISA plates (Nunc) were coated with 5 μg/ml D. farinae at 4°C overnight. Plates were then blocked with 1% Bovine Serum Albumin (BSA) in PBS for at least 2 hours at room temperature. Sera diluted in PBS containing 1% BSA were added and incubated in the blocked wells for 2 hours at 37°C. Afterwards, biotinylated detection antibodies (rat anti-mouse IgG1 and rat anti-mouse IgE; BD Biosciences; 1:1000) were then added for 1 hour at room temperature followed by incubation with horseradish peroxidase-conjugated streptavidin (BD Biosciences; 1:2000) for 30 minutes at room temperature. Reaction was then revealed using supersensitive 3,3',5,5'-tetramethylbenzidine (TMB; Sigma Aldrich) substrate and OD was measured at 450 nm.

D. farinae samples were diluted to 200 μg/mL in a reaction buffer composed of Sodium
Phosphate 100 mM pH 6, EDTA 10 mM, and DTT 1 mM. The extract was incubated or not with irreversible protease inhibitors (cysteine protease inhibitor E64 100 nM or serine protease inhibitor AEBSF 1 mM) in a reaction volume of 50 μL. Activity was measured using Boc-Gln-Ala-Arg-AMC fluorogenic substrate (200 μM, diluted in the same reaction buffer). D. farinae extracts and substrate solutions were incubated separately for 20 minutes at 37°C. Immediately after addition of 50 μL substrate solution on the D. farinae extracts, the release of AMC fluorescence was followed over time using a varioskan Flash apparatus (Thermo Scientific). The initial velocity reflecting proteolytic activity was calculated from the kinetics curves. A control was performed by heat-inactivating D. farinae extract for 60 minutes at 95°C before addition of substrate. An additional control was performed in a reaction buffer devoid of DTT.

Dorsal Root Ganglia (DRG) dissociation, culture, and Ca 2+ imaging
DRG neurons from all spinal levels were collected in ice-cold Dulbecco's modified Eagle's medium (DMEM)/F-12 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). DRGs were digested with a mixture of dispase (5 mg/ml) and collagenase type I (1 mg/ml) enzyme at 37°C for 45 minutes. After dissociation, cells were spun at 300g and re-suspended in media before being plated on glass coverslips coated with poly-D-lysine (0.5 mg/ml) and laminin (10 μg/ml, Invitrogen) or Lab-tek II chambers slide with cover (Thermo Fisher Scientific). DRGs were cultured in media supplemented with 50 ng/mL NGF at 37°C overnight (12-24 hours) before experimentation. In some experiments, control peptide, or MyD88 inhibitory peptide (100 μM), were added to the DRG cultures for 16 h. Cells were imaged in calcium imaging buffer (CIB; 10 mM HEPES, 1.

Quantification of SP secretion
DRG of mice were cultured as described above, rinsed in Hanks Balanced Salt Solution (HBSS; Sigma), and incubated in L-cysteine containing 1% Papain (Sigma) for 10 minutes at 37°C. After a wash with filtered Leibovitz's L-15 Medium (Gibco) containing 10% FBS (Sigma), they were incubated in HBSS containing collagenase (1 mg/ml; Sigma) and dispase (4 mg/ml, Sigma) at 37°C for 10 min. After titration, cells were plated in a Poly-D-lysinelaminin (Sigma)-coated 12 well-plate (Corning) and recovered with the complete culture media (DMEM-Glutamax, 2.5% FBS, 1% pen/strep and ARAC, FUDR, uridine -10 μM each; all from Sigma). Cells were incubated for 5 minutes with vehicle or 5 ng/ml D. farinae or 5 ng/ml SEB or and 125 nM capsaicin. Cell supernatants were tested with a Substance P Elisa kit (Enzo) following the manufacturer's recommendations. High resolution Z-stack images 1024 x 1024 pixels of Av.SRho fluorescent signal were acquired using a 20x objective. Images were then processed using Image J and Imaris (Bitplane) softwares.

In vivo two-photon microscopy of living mice
Experiments were conducted as previously described 25,37  . Modeling and analysis of fluorescent signals were performed using untreated image sequences, as previously described 25 , using Imaris software (Bitplane) and Image J software version Fiji, respectively.

Automated computational analysis of the minimum distance between mast cells and neurons in the dermis of living mice
1) 3D high resolution images were taken using a Prairie Ultima IV two-photon microscope as described above. The following steps have been automated in the software Imaris Bitplane version 9.2. 2) Hair follicles' autofluorescent signals were modeled into matched 3D objects using the isosurface algorithm. 3) Autofluorescent signals corresponding to the generated isosurfaces were depleted from the GCaMP3 fluorescence detection channel so that the hair follicles were no longer detectable in that particular channel. 4) The filament tracer algorithm was applied in the GCaMP3 fluorescence detection channel in order to precisely trace the trajectories and exact shapes of GCaMP3 fluorescent signals. Filament traces were then converted into fluorescent signals in a new fluorescent channel. 5) Those newly generated fluorescent signals were modeled into matched 3D objects using the isosurface algorithm. The distance transformation algorithm was applied to those new isosurfaces resulting in the generation of a new distance transformation channel. 6) Av.SRho fluorescent signals were modeled into matched 3D objects using the isosurface algorithm. The intensity minimum (i.e., distance minimum in μm) to the distance transformation channel (i.e., modeled sensory neurons) was calculated for each of the Av.SRho + isosurfaces of at least 5 μm of diameter (corresponding to Av.SRho + mast cell cellular bodies and excluding small exteriorized Av.SRho + granules structures). 7) Results per field of view were generated into separated Excel sheets. The exact same procedure was automatically applied to all analyzed 3D images.

Statistics
Statistical tests were performed with the software Prism 6 (GraphPad Software). Two-tailed unpaired/paired Student's t tests, 1-way ANOVA with Tukey's test for multiple comparisons or Holm-Sidak's post-hoc test were performed on samples as noted in the respective figure legends. A P value of less than 0.05 was considered statistically significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Nat Immunol. Author manuscript; available in PMC 2020 April 07.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts computational analysis of the minimum distance between modeled Av.SRho + mast cell bodies detected and modeled capsaicin-activated TRPV1 + Pirt-GCaMP3 + sensory neurons. e, Examples of modeled Av.SRho + mast cell bodies forming physical contact with (left) or in proximity to (right) modeled capsaicin-activated TRPV1 + Pirt-GCaMP3 + sensory neurons. f, Proportion (%) of 453 modeled Av.SRho + mast cell bodies from the ear pinnae of 3 different mice in physical contact with (red), at 1 to 25 μm (black), at 25 to 50 μm (dark grey) or at more than 50 μm (light grey) from modeled capsaicin-activated TRPV1 + Pirt-GCaMP3 + sensory neurons.