IL-33 signaling in sensory neurons promotes dry skin itch

Abstract

Background: Chronic pruritus, or itch, is common and debilitating, but the neuroimmune mechanisms that drive chronic itch are only starting to be elucidated. Recent studies demonstrate that the IL-33 receptor (IL-33R) is expressed by sensory neurons. However, whether sensory neuron-restricted activity of IL-33 is necessary for chronic itch remains poorly understood.

Objectives: We sought to determine if IL-33 signaling in sensory neurons is critical for the development of chronic itch in 2 divergent pruritic disease models.

Methods: Plasma levels of IL-33 were assessed in patients with atopic dermatitis (AD) and chronic pruritus of unknown origin (CPUO). Mice were generated to conditionally delete IL-33R from sensory neurons. The contribution of neuronal IL-33R signaling to chronic itch development was tested in mouse models that recapitulate key pathologic features of AD and CPUO, respectively.

Results: IL-33 was elevated in both AD and CPUO as well as their respective mouse models. While neuron-restricted IL-33R signaling was dispensable for itch in AD-like disease, it was required for the development of dry skin itch in a mouse model that mirrors key aspects of CPUO pathology.

Conclusions: These data highlight how IL-33 may be a predominant mediator of itch in certain contexts, depending on the tissue microenvironment. Further, this study provides insight into future therapeutic strategies targeting the IL-33 pathway for chronic itch.

Keywords: Atopic dermatitis; IL-33; chronic pruritus of unknown origin; dry skin; itch; neuroimmunology; pruriceptor; pruritogen.

Figure 1:. Mouse and human DRG express IL-33R.

(A) Gel of Il1rl1 RT-PCR product from dorsal root ganglia (DRG) isolated from one wild-type (WT) mouse. Representative of three mice. (B) Representative calcium imaging trace of mouse DRG neuron in response to vehicle (Veh), recombinant mouse (rm)IL-33, capsaicin (Cap), and potassium chloride (KCl). (C) Percent of rmIL-33-, histamine (His)-, and Cap-responsive DRG neurons out of all KCl-responsive DRG neurons. (D) Venn diagrams of overlapping responses between IL-33-responsive (IL-33⁺) and Cap-responsive (Cap⁺) or His-responsive (His⁺) neurons. (B-D) n > 900 neurons from at least 4 WT mice (6 combined experiments). (E) Gel of IL1RL1 RT-PCR product from DRG isolated from one human donor. Representative of three donors. *Ladder has been previously published in Oetjen et al. (F) Representative calcium imaging trace of human DRG neuron in response to Veh, recombinant human (rh)IL-33, Cap, and KCl. (G) Percent of rhIL-33- and Cap-responsive DRG neurons out of all KCl-responsive DRG neurons. (H) Venn diagrams of overlapping responses between IL-33⁺ and Cap⁺ neurons. (F-H) N > 200 neurons from 2 human subjects (2 combined experiments). (I) t-SNE plot of single cell RNA-seq of mouse DRG colored by cell populations. Violin plots of (J) Il1rl1 and (K) Il4ra gene expression. Full dataset in Avraham et al.

Figure 2:. Generation of IL-33R conditional deletion mice

(A) Map of Il1rl1 conditional knock-out allele. blue triangles, loxP sites; gray boxes, exons; red box, conditionally deleted region. (B) Expression of Il1rl1 in lymph node-derived immune cells (left) and MACS-sorted sensory neurons (right) from littermate (LM) control and IL-33R^Δneuron mice by RT-qPCR. n > 3 mice/group. (C-G) Assessment of (C) motor activity (rotarod), (D) thermal pain behavior (hot plate), and acute itch behavior following intradermal injection (i.d.) of (E) histamine, (F) chloroquine, and (G) serotonin in LM control and IL-33R^Δneuron mice. (C-G) n > 4 mice/group (E-G), 2 combined experiments. Not significant (NS), *p<0.05 by unpaired, two-tailed t test.

Figure 3:. IL-33R signaling in sensory neurons is dispensable for chronic itch in AD-like disease.

(A) Schematic of the measurement of IL-33 in the plasma of 11 healthy control (HC) subjects and 11 patients with atopic dermatitis (AD) by Luminex multiplex ELISA. (B) Amount of IL-33 in the plasma of HC subjects and patients with AD. (C) Representative clinical images and H&E sections of ear skin from ethanol (EtOH)- or MC903-treated WT mice (day 12). Scale bar is 50 μm. (D) Heatmap and hierarchical clustering of significantly differentially expressed genes in the ear skin of EtOH- or MC903-treated WT mice (day 12). The most differentially expressed genes (1,300 genes) are displayed (based on the t statistic value). n = 4 mice/group. Full dataset in Oetjen et al. (E) Representative clinical images and H&E sections of MC903-treated LM control and IL-33R^Δneuron mice (day 12). Scale bar is 20 μm. (F) Percent change in ear thickness and (G) number of scratching bouts from MC903-treated LM control and IL-33R^Δneuron mice over time (days). n = 13–18 mice/group (2–3 combined experiments). (B) *p<0.05 by unpaired, two-tailed t test. (F-G) NS by multiple t test using Holm-Sidak method.

Figure 4:. IL-33 is elevated in CPUO.

Representative clinical images from a patient with (A) AD and (B) chronic pruritus of unknown origin (CPUO). Black boxes indicate zoomed-in view of skin. Representative H&E skin sections from (C) control, (D) patient with AD, and (E) patient with CPUO. Bracket, stratum corneum (SC); brace, epidermis (Epi); black arrow, spongiosis; gray arrow, vascular dilatation; white arrow, dermal perivascular immune infiltrate. Scale bar represents 100 μm. (F) Schematic of the measurement of IL-33 in the plasma of 11 HC subjects (same subjects as in Figure 1) and 8 patients with CPUO by Luminex multiplex ELISA. (G) Amount of IL-33 in the plasma of HC subjects and patients with CPUO. *p<0.05 by unpaired, two-tailed t test.

Figure 5:. Dry skin itch is dependent on IL-33 signaling in sensory neurons.

(A) Representative clinical images of skin from water- or AEW-treated WT mice (day 5). Frequency of (B) immune cells (C) mast cells and (D) group 2 innate lymphoid cells (ILC2s) in the skin of water- or AEW-treated WT mice (day 5). n = 6–8 mice/group (2 combined experiments). (E) Number of scratching bouts from water- or AEW-treated WT mice (day 5). n = 5 mice/group (representative of 3 experiments). (F) Expression of Il33 by RT-qPCR in water- or AEW-treated skin of WT mice (day 4). n = 5–7 mice/group (2 combined experiments). Number of scratching bouts from AEW-treated (G) LM control and MasTRECK mice, (H) control (Cont) and Rag2/Il2rg^−/− mice, (I) LM control and IL-33R^Δimmune mice, and (J) LM control and IL-33R^Δneuron mice (day 5). (G-J) n = 9–18 mice/group (2 combined experiments). (K) Number of scratching bouts following i.d. injection of Veh or rmIL-33 in WT mice. n = 6–8 mice/group (2 combined experiments). (L) Representative calcium traces of mouse DRG neurons responding to chloroquine (CQ) after exposure to Veh or rmIL-33. Each trace represents one neuron. (M) Percent of CQ-responsive neurons out of all KCl-responsive neurons following exposure to Veh or rmIL-33. n = >400 neurons from 3 mice (2 combined experiments). (B-J, M) NS, *p<0.05, **p<0.01, ****p<0.0001 by unpaired, two-tailed t test. (K) NS by one-way ANOVA with multiple comparisons.