Scratching promotes allergic inflammation and host defense via neurogenic mast cell activation

Abstract

Itch is a dominant symptom in dermatitis, and scratching promotes cutaneous inflammation, thereby worsening disease. However, the mechanisms through which scratching exacerbates inflammation and whether scratching provides benefit to the host are largely unknown. We found that scratching was required for skin inflammation in mouse models dependent on FcεRI-mediated mast cell activation. Scratching-induced inflammation required pain-sensing nociceptors, the neuropeptide substance P, and the mast cell receptor MrgprB2. Scratching also increased cutaneous inflammation and augmented host defense to superficial Staphylococcus aureus infection. Thus, through the activation of nociceptor-driven neuroinflammation, scratching both exacerbated allergic skin disease and provided protection from S. aureus, reconciling the seemingly paradoxical role of scratching as a pathological process and evolutionary adaptation.

Fig. 1.. MrgprA3-expressing neurons and scratching are required for FITC and Ox CHS.

(A to C) DT-treated Mrgpra3^DTR (red circles) or LMC (black circles) mice were sensitized on shaved abdominal skin with FITC (LMC and Mrgpra3^DTR n = 7 mice) (A), Ox (LMC and Mrgpra3^DTR n = 10) (B), or DNFB (LMC and Mrgpra3^DTR n = 27) (C) hapten followed by challenge 5 days later on the ear with the same hapten. Ear thickness at the indicated time points after challenge is shown (FITC LMC and Mrgpra3^DTR n = 7; Ox LMC and Mrgpra3^DTR n = 10; DNFB LMC n = 9 and DNFB Mrgpra3^DTR n = 8). (D) The number of scratching bouts observed over 30 min in mice 1 day postchallenge, with the indicated hapten shown (LMC n = 8 to 9, Mrgpra3^DTR n = 7 to 10). (E) Ear thickness 1 day after hapten challenge in DT-treated LMC and Mrgpra3^DTR mice as well as Elizabethan-collared control mice (LMC collar; black triangles) is shown (FITC, LMC n = 9, Mrgpra3^DTR n = 15, collar n = 6; Ox, LMC n = 20, Mrgpra3^DTR n = 15, collar n = 5; DNFB, LMC and Mrgpra3^DTR n = 31, collar n = 17). Results in (A) to (C) are represented as means ± SEMs from three to five independent experiments. Individual data points in (D) and (E) represent data from a single animal, and bars are means ± SEMs from three to five independent experiments. Significance was calculated using unpaired Student’s t test [(A) to (C)], Mann-Whitney (D), or one-way analysis of variance (ANOVA) with multiple comparisons (E). *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.

Fig. 2.. Scratching is required for neutrophilic infiltrate and mast cell activation.

(A) Representative H&E sections of ears from sensitized DT-treated LMC (black circles), Mrgpra3^DTR (red circles), and collared LMC (black triangles) mice 24 hours after FITC or Ox challenge. (B) The total number of neutrophils from untreated mice, FITC-challenged DT-treated LMC, Mrgpra3^DTR, and collared LMC mice at 24 hours, as determined by flow cytometry (untreated n = 4, LMC and Mrgpra3^DTR n = 11, collar n = 5). (C) Same as in (B) but sensitized and challenged with Ox (untreated n = 3, LMC n = 9, Mrgpra3^DTR n = 5, collar n = 6). (D and E) Relative expression of Tnf mRNA based on quantitative reverse transcription PCR (RTqPCR) of whole ear skin from unmanipulated, Mrgpra3^DTR and collared LMC mice 24 hours after FITC (untreated n = 4, LMC n = 8, Mrgpra3^DTR and collar n = 6) (D) and Ox challenge (untreated n = 4, LMC and Mrgpra3^DTR n = 8, collar n = 5) (E). (F and G) Ear thickness at the indicated time points after FITC (LMC n = 17, Mrgpra3^DTR n = 18, collar n = 11) (F) and Ox (LMC n = 36, Mrgpra3^DTR n = 20, collar n = 10) (G) challenge is shown. (H) Quantification of EB dye extravasation in FITC- or Ox-sensitized DT-treated LMC, Mrgpra3^DTR, and collared LMC mice 10 hours after FITC challenge and 12 hours after Ox challenge (LMC n = 7, Mrgpra3^DTR and collar n = 5). (I) Immunofluorescent microscopic visualization of ear skin 10 hours after FITC challenge illustrates avidin+ mast cells (red) and DAPI nuclear label (blue) in DT-treated LMC, Mrgpra3^DTR, and LMC collared mice. Background FITC appears green. Regions highlighted by dotted lines are shown at higher magnification in insets. (J) Quantification of the number of degranulated mast cells observed in (I) is shown (LMC and Mrgpra3^DTR n = 8, collar n = 5). Scale bars [(A) and (I)], 200 μm. Individual data points represent data from a single animal, and bars show means ± SEMs from three independent experiments [(B) to (E), (H), and (J)]. Results in (F) and (G) are represented as means ± SEMs from three independent experiments. Significance was calculated using a one-way ANOVA with multiple comparisons [(B) to (H) and (J)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. Scratching is required for neutrophilic inflammation after FcεRI-mediated mast cell activation.

(A) The number of scratching bouts per hour in DT-treated LMC (black circles) and Mrgpra3^DTR (red circles) at the indicated time after i.d. injection of 1.2 μg of SP (“SubP”) into the ear is shown. (B) Ear thickness in DT-treated LMC, Mrgpra3^DTR, and collared LMC (black triangles) mice at the indicated time after i.d. injection of SP into the ear is shown (LMC n = 9, Mrgpra3^DTR n = 5, collar n = 4). (C and D) Same as in (A) and (B) except mice were sensitized with 20 ng of dinitrophenyl-specific IgE followed by i.d. injection of 2 μg of DNP to the ear pinna 20 hours later (LMC n = 10 to 15, Mrgpra3^DTR n = 7 to 11, collar n = 6). (E) Quantification of EB dye extravasation 10 hours after DNP challenge (LMC n = 8, Mrgpra3^DTR n = 6, collar n = 5). (F) Immunofluorescent microscopic visualization with DAPI (blue) and anti-Gr1 (magenta) to visualize neutrophils in ear skin 10 hours after DNP challenge. (G) Quantification of numbers of neutrophils in (F) is shown (LMC n = 7, Mrgpra3^DTR n = 6, collar n = 5). (H and I) Expression of normalized Tnf mRNA (H) and TNF protein expression (I) in whole ear skin from unmanipulated mice (black squares) and DT-treated LMC, Mrgpra3^DTR, and LMC collared mice 10 hours after DNP challenge (untreated n = 4; LMC, Mrgpra3^DTR, and collar n = 5). Scale bar (F), 200 μm. Results in (A) to (D) are represented as means ± SEMs from three independent experiments. Individual data points [(E) and (G) to (I)] represent data from a single animal, and bars show means ± SEMs from three independent experiments. Significance was calculated using a Mann-Whitney test [(A) and (C)] or by one-way ANOVA with multiple comparisons [(B), (D), (E), and (G) to (I)]. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.. Scratching-induced inflammation requires neurogenic mast cell activation.

(A) WT (black circles), collared WT (black triangles), and TrpV1^hM4Di (blue triangles) mice were challenged with DNP and quantification of SP in supernatants from ex vivo skin explant organ cultures of ear tissue harvested 30 min after challenge. Unchallenged (black squares) and collared mice treated with capsaicin (open squares) are also shown (unchallenged, capsaicin, and DNP collar n = 8; DNP n = 16; TrpV1^hM4Di n = 6). (B and C) Percentage of mast cell degranulation as calculated by β-hex release (B) and TNF protein levels (in picograms per milliliter) (C) from the culture supernatants of DNP-specific IgE-sensitized cultured PMCs from six mice across three experiments 6 hours after treatment with the indicated dose of DNP and 48/80. (D to F) Ear thickness (D), quantification of EB extravasation (E), and Tnf mRNA (F) measured at 10 hours after DNP challenge in vehicle-treated (black circles), QWF-treated (magenta circles), MrgprB2^−/− (purple circles), and Tac1^−/− (lavender circles) DNP-specific IgE-sensitized mice. (G) Scratching behavior over 30 min after DNP challenge in vehicle-treated, QWF-treated, MrgprB2^−/−, and Tac1^−/− mice (vehicle n =9 to 13, QWF n = 7, MrgprB2^−/− n = 8 to 12, Tac1^−/− n = 9). (H to J) Ear thickness (H), quantification of EB extravasation (I), and Tnf mRNA (J) measured at 10 hours after DNP challenge in CNO-treated LMC and TrpV1^hM4Di mice (LMC n =7 to 10, TrpV1^hM4Di n =8 to 9). (K) Scratching behavior over 30 min after DNP challenge. (L to N) Ear thickness (L), quantification of EB extravasation (M), and Tnf mRNA (N) measured at 10 hours after capsaicin treatment (black squares), DNP challenge (black circles), DNP challenge in collared mice (black triangles), and DNP and capsaicin in collared mice (green squares) (capsaicin, DNP, and DNP capsaicin collar n = 8; DNP collar n = 7). (O to Q) Ear thickness (O), quantification of EB extravasation (P), and degranulated mast cells (Q) quantified at 10 hours after FITC challenge in cohorts, as in (L) to (N) (capsaicin n =7 to 8, FITC n =7 to 9, collar n = 6 to 7, FITC capsaicin collar n = 8). Individual data points represent data from a single animal, and bars show means ± SEMs from three independent experiments. Significance was calculated using a one-way ANOVA with multiple comparisons [(A), (D) to (G), and (L) to (Q)], unpaired Student’s t test [(H) to (J)], or Mann-Whitney test (K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.. Scratching is required for S. aureus–induced scratching and host defense.

(A and B) Shannon alpha diversity (A) and linear discriminant analysis (LDA) (B) of 16S rRNA-seq of the indicated groups. (C) The identity of live genera isolated from ear swabs obtained 24 hours after FITC challenge of the indicated group is shown (LMC and Mrgpra3^DTR n = 5, collar n = 6). (D) Scratching behavior over 30 min after i.d. nape injection of Staph V8 protease in DT-treated LMC and Mrgpra3^DTR mice (LMC and Mrgpra3^DTR n = 8). (E) Scratching behavior over 30 min immediately after removal of occlusive S. aureus patch in DT-treated LMC (black circles) and Mrgpra3^DTR (red circles) mice (LMC and Mrgpra3^DTR n = 9). (F) Ten hours after patch removal, alloknesis was measured by a 0.04g von-Frey filament out of nine total stimuli in DT-treated LMC and Mrgpra3^DTR mice (LMC n = 8 and Mrgpra3^DTR n = 9). (G) Scratching behavior over 30 min after epicutaneous S. aureus ear infection in naïve and sensitized DT-treated LMC and Mrgpra3^DTR mice (LMC n = 9 to 11 and Mrgpra3^DTR n = 8 to 10). (H to K) Ear thickness (H), quantification of EB extravasation (I), Tnf mRNA (J), and degranulated mast cells (K) quantified at 8 hours after epicutaneous S. aureus ear infection in sensitized DT-treated LMC, Mrgpra3^DTR, and LMC collared (black triangles) mice (LMC n = 8 to 14, Mrgpra3^DTR n = 5 to 8, collar n = 6 to 10). (L) S. aureus ear CFU in DT-treated LMC, Mrgpra3^DTR, and LMC collared mice that were either previously uninfected (naïve) or previously sensitized (LMC n =4 to 14, Mrgpra3^DTR n = 4 to 10, collar n = 5 to 9). Tissue was harvested 24 hours after epicutaneous ear infection. Bars in (A) to (C) represent 25th to 75th percentiles, with whiskers extending to 10th and 90th percentiles. Horizontal bars indicate the medians. Individual data points in (D) to (L) represent data from a single animal, and bars show means ± SEMs from three independent experiments. Significance was calculated using a Student’s paired t test (A), a Mann-Whitney test [(D) to (G)], or a one-way ANOVA with multiple comparisons [(H) to (L)]. *P < 0.05; **P < 0.01; ***P < 0.001.