IL-33 potentiates histaminergic itch

Abstract

Background: Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined.

Objectives: Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch.

Methods: Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls.

Results: IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls.

Conclusions: Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

Keywords: Chronic spontaneous urticaria; IL-13; IL-33; histamine; itch; mast cell; neuroimmunology.

Figure 1.. IL-33 enhances histaminergic itch.

(A) Number of scratching bouts following intradermal (i.d.) co-injection of histamine and IL-33 (20 μg histamine + 300 ng IL-33 in 20 μL) or histamine alone (20 μg in 20 μL) in wild-type (WT) mice. (B) Number of scratching bouts following i.d. injection of IL-33 (300 ng in 20 μL) or saline control and i.d. injection of histamine (20 μg in 20 μL) 6 hours later in WT mice. (C) Number of scratching bouts 6 hours after i.d. injection of IL-33 (300 ng in 20 μL) or saline control. Number of scratching bouts following IL-33 pretreatment and subsequent histamine challenge in (D) IL-33R^∆neuron (Na_V1.8^Cre x IL-33^fl/fl) and littermate (LM) mice or (E) IL-33R^∆immune (Vav^Cre x IL-33^fl/fl) and LM mice. (A-E) n = 6–10 mice/group with two combined independent experiments/figure. Not significant (NS), *p<0.05, ***p<0.001 by unpaired, two-tailed t test. Data are represented as mean ± SD.

Figure 2.. Mast cells are top expressors of IL-33R and mediate IL-33-enhanced histaminergic itch.

(A) UMAP visualization shows 17 cell clusters in mouse skin (GSE149121, n = 3, clusters 5 and 9 omitted – see methods). (B) Dot plot of Cpa3, Cma1, and Il1rl1 gene expression across clusters in (A). (C) Number of scratching bouts following IL-33 or saline control pretreatment and subsequent OVA challenge (i.d., 50 μg) in WT mice. Mice were sensitized the day prior with anti-ovalbumin IgE (αOVA IgE; 2 μg in 100 μL intravenously, i.v.). (D) Number of scratching bouts following saline vehicle control or IL-33 pretreatment and subsequent challenge with compound 48/80 (C48/80) in WT mice. (E) Number of scratching bouts following IL-33 pretreatment and subsequent histamine challenge in Sas^−/− and LM mice. (C-E) n = 5–10 mice/group with two combined independent experiments/figure. *p<0.05 by unpaired, two-tailed t test. Data are represented as mean ± SD.

Figure 3.. IL-13 is induced by IL-33 in mast cells and mediates IL-33-enhanced histaminergic itch.

Volcano plots of differential gene expression from microarrays of bone marrow-derived mast cells (BMMCs) after (A) acute - 4 hours (GSE96695) and (B) chronic - 4–6 weeks (GSE39382) treatment with IL-33 vs control. Key pruritogenic cytokines are indicated. Bonferroni adjusted p value (false discovery rate – FDR) cutoff 0.05, log2 fold change cutoffs <−1 and >1. (C) Overlap of significantly upregulated (FDR < 0.05) genes between treatment and control for acute (4 hour) and chronic (4–6 weeks) IL-33 treatment of BMMCs. Gene expression heatmaps of top 100 genes upregulated compared to control only in BMMCs treated with IL-33 for (D) 4 hours or (E) 4–6 weeks. (F) Gene expression heatmaps of top 20 genes upregulated by both acute and chronic IL-33 treatment of BMMCs. (G) Number of scratching bouts following IL-33 or saline control pretreatment and subsequent histamine challenge in Il13^−/− and WT mice. (G) n = 5–10 mice/group with two combined independent experiments/figure. *p<0.05 by unpaired, two-tailed t test. Data are represented as mean ± SD.

Figure 4.. IL-33R is highly expressed in human skin mast cells and increased in skin from subjects with CSU.

(A) UMAP visualization shows 40 cell clusters in human skin (E-MTAB-8142, N = 5). (B) Dot plot of CPA3, CMA1, and IL1RL1 gene expression across clusters. Gene expression heatmaps of all differentially expressed genes (Bonferroni adjusted p value cutoff < 0.05) in microarrays of skin biopsies from lesional and non-lesional skin from subjects with chronic spontaneous urticaria (CSU) and from healthy controls in two different study cohorts for (C) cohort 1 (GSE72450) and (D) cohort 2 (GSE57178).