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 on 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 following IL-33 or saline administration. Various stimuli and conditional and global knock-out mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic datasets 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 biopsies 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 (IL-33R) 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-33R 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.

Graphical Abstract

CAPSULE SUMMARY:

IL-33 stimulates a mast cell- and IL-13-dependent axis to exacerbate of histaminergic itch. These findings provide insight into mast cell-associated itch conditions such as chronic spontaneous urticaria.

INTRODUCTION

Histamine was one of the first molecules identified to act as a pruritogen. Antihistamines are often a first line therapy for pruritic conditions, yet many patients have minimal response or develop refractory symptoms over time.^1,2 Mechanisms underlying the limited efficacy of antihistamines in pruritic disorders remain poorly understood.

The alarmin cytokine IL-33 is elevated in chronic pruritic conditions such as atopic dermatitis, chronic spontaneous urticaria (CSU), and dry skin itch.^3–5 Although the role of IL-33 in itch is only beginning to be elucidated, recent evidence suggests that IL-33 may drive chronic itch development by inducing peripheral itch sensitization. Indeed, we and others have found that signaling of the IL-33 receptor (IL-33R) in sensory neurons is required for chronic itch in certain mouse models.4,6 We hypothesized that IL-33 may augment histaminergic itch through neuromodulatory mechanisms to limit effectiveness of antihistamines in chronic pruritic conditions. Instead, we found IL-33 can promote itch through a novel pathway involving mast cells and IL-13.

RESULTS AND DISCUSSION

We and others have shown that IL-33 does not act like a classic pruritogen as intradermal (i.d.) injection of IL-33 does not induce an immediate acute itch response.^4,6 Thus, we hypothesized that IL-33 may instead promote itch by enhancing neuronal responsiveness to pruritogens like histamine, similar to IL-4 and oncostatin M (OSM).^7,8 However, in contrast to IL-4 and OSM, we observed no difference in acute scratching (itch behavioral response) between wildtype (WT) mice that received intradermal (i.d.) co-injection of IL-33 and histamine or histamine alone (Fig 1, A). Instead, IL-33 significantly increased histamine-provoked itch behavior if mice were pretreated with IL-33 six hours prior to histamine injection (Fig 1, B). This effect was not due to IL-33 directly provoking itch, since we observed no difference in scratching six hours after mice were i.d. injected with IL-33 or saline control (in absence of subsequent histamine challenge) (Fig 1, C). We next examined whether potentiation of histaminergic itch by IL-33 was mediated by neuronal IL-33R signaling as we initially hypothesized. We utilized a mouse strain in which IL-33R was conditionally deleted from sensory neurons (IL-33R^∆neuron).⁴ In contrast to our hypothesis, we found no difference in itch behavior between IL-33R^∆neuron and littermate (LM) control mice in the IL-33 pretreatment and histamine challenge (IL-33 + His) model (Fig 1, D). These findings suggest that IL-33 magnifies histaminergic itch via an intermediary cell type, rather than through direct effects on sensory neurons, in contrast to mechanisms identified for other itch-promoting cytokines like IL-4 or OSM.^7,8

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.

As an alarmin, a canonical function of IL-33 is its ability to rapidly activate innate immune cells to mobilize a type 2 immune response.⁹ However, whether this axis plays a role in itch remains unknown. Thus, to test if IL-33R signaling in immune cells is required for its ability to amplify histaminergic itch, we utilized mice that conditionally lack IL-33R expression in immune cells (IL-33R^∆immune).⁴ Strikingly, scratching in the IL-33 + His model was significantly decreased in IL-33R^∆immune mice compared to LM control mice (Fig 1, E).

To determine which immune cells may be responding to IL-33, we utilized the Immunological Genome Project (ImmGen) to analyze the expression level of Il1rl1 (IL-33R) across different immune cell populations from a variety of tissues (see online repository). In support of prior studies,⁹ several mast cell populations were among the cell types with the highest expression levels of IL-33R (Fig E1, A and B). To evaluate IL-33R expression specifically within skin tissue, we reanalyzed a previously published single-cell RNA sequencing (scRNA-seq) dataset of mouse skin containing 17 different mouse cell types (Fig 2, A).¹⁰ Consistent with the ImmGen data, mast cells (cluster 12) had the highest expression of Il1rl1 across the 17 identified cell clusters (Fig 2, B). However, despite the high of expression of IL-33R on mast cells, how IL-33 impacts mast cell function remains a highly active area of investigation.

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.

To test the potential role of mast cells in IL-33-enhanced histaminergic itch, we first used two mast cell-specific reagents known to induce histaminergic itch through orthogonal pathways. A classic model of mast cell activation involves the passive transfer of anti-ovalbumin (OVA) IgE by intravenous (i.v.) injection followed by i.d. OVA challenge the following day. We recently showed that this kind of antigen-mediated itch is highly histaminergic in nature.^11,12 Alternatively, compound 48/80 (C48/80) induces degranulation by activating the mast cell-specific G protein coupled receptor Mrgprb2.¹³ When we administered i.d. IL-33 or saline control in WT OVA-sensitized mice 6 hours prior to i.d. OVA challenge, we observed that mice treated with IL-33 had enhanced itch behavior compared to those that received saline control (Fig 2, C). Similarly, we found that mice pretreated with IL-33 had enhanced itch behavior compared to those that received saline control when challenged 6 hours later with i.d. C48/80 (Fig 2, D). Next, we utilized Sas^−/− mice, which developmentally lack mast cells as their primary phenotype.¹⁴ In our IL-33 + His model, Sash^−/− mice scratched significantly less compared to LM control mice (Fig 2, E). Taken together, our findings demonstrate a central role of mast cells in mediating IL-33-enhanced histaminergic itch.

We next sought to investigate potential effector molecules that may promote IL-33 enhancement of histaminergic itch. Prior studies, largely in vitro, have shown that IL-33 can modulate mast cell mediator release in response to activating stimuli.^15–17 To examine whether IL-33 stimulation modulates expression of pruritogenic pathways, we reanalyzed two previously published microarrays of mast cells to determine differentially expressed genes after acute or chronic treatment with IL-33 (Fig. 3, A and B).^18,19 We found distinct, but overlapping, transcriptional profiles of upregulated genes depending on duration of IL-33 treatment (Fig. 3, C). Interestingly, only acute IL-33 stimulation induced expression of its own receptor, Il1rl1 (Fig. 3, D), while chronic stimulation led to increased expression of the histamine receptor Hrh1 (Fig. 3, E), suggesting mast cell responses to IL-33 are dynamic. Among 320 genes upregulated by both acute and chronic IL-33 stimulation was tryptase (Tpsab1) (Fig. 3, F), which can potentially cleave IL-33 into more potent forms to further drive activation of this circuit.²⁰ Critically, of known pruritogenic cytokines, only IL-13 (Il13) was significantly upregulated by both acute and chronic IL-33 treatment (Fig. 3, A, B, and F), in agreement with prior studies.^15–17 Indeed, IL-13 can directly activate sensory neurons and induce neuronal sensitization to various pruritogens.^7,21 Thus, we hypothesized that IL-13 is a key mediator of IL-33-enhanced histaminergic itch. To test this, we utilized IL-13 knockout mice and observed significantly less scratching in Il13^−/− mice compared to WT mice in the IL-33 + His model (Fig 3, G). While additional studies are required to further dissect this axis, and other immune cells and/or pruritogenic cytokines like IL-31 may also be involved, our findings indicate that mast cells and IL-13 are critical mediators of IL-33-enhanced histaminergic itch.

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.

Increasing evidence suggests that IL-33 can potentiate mast cell-mediated inflammation.^15–17 Yet, the significance of this axis in itch has been largely unexplored. Our findings suggest that IL-33 augments histaminergic itch through mast cell activation and IL-13 production. While IL-33 signaling on sensory neurons modulates non-histaminergic dry skin itch,⁴ we found it was dispensable in our model of histaminergic itch. This discrepancy may arise from an indirect immune axis overriding the direct neuronal axis or distinct downstream effects of IL-33R activation on modulating activity of pruritogen receptors.

Our observations have important implications for clinical use of antihistamines that warrant further investigation. For example, mast cells are recognized as critical pathogenic mediators of (CSU), a disorder characterized by the development of itchy hives that last longer than six weeks.²² The only FDA-approved treatments available for CSU are high dose antihistamines (first-line) and the anti-IgE monoclonal antibody (mAb) omalizumab (second-line).22 Yet, despite the central role mast cell-derived histamine plays in the pathogenesis of urticaria, up to 50% of CSU patients continue to have symptoms despite maximal on-label therapy.^1,22

Given our observations suggesting that IL-33 can expand the pruritogenic capacity of histamine in mice, we wondered if similar mechanisms may contribute to CSU pathogenesis in humans. To test this, we first analyzed a published scRNA-seq dataset of healthy human skin consisting of 40 previously identified cellular subtypes (Fig 4, A).²³ Consistent with our findings in mice, mast cells (Cluster 19, identified by high expression of CPA3 and CMA1) most highly expressed IL1RL1 (IL-33R) in human skin (Fig 4, B). Next, we examined differentially expressed genes in two published microarray datasets of skin biopsies from healthy controls and subjects with CSU.^24,25 Notwithstanding the limitations of bulk transcriptional analysis to assign differences in gene expression to specific cell types, we found IL1RL1 and HRH1 (histamine receptor 1 H1) were significantly upregulated in lesional CSU skin compared to healthy controls in both datasets (Fig. 4, C and D).

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).

These findings suggest skin in CSU is primed to respond to IL-33 and histamine and provide a mechanism by which IL-33-enhanced histaminergic itch may contribute to symptoms in CSU. Furthermore, the ability of IL-33 to expand the pruritogenic capacity of histamine may explain the poor responses to antihistamines not only in CSU but also in other pruritic conditions traditionally considered non-histaminergic such as atopic dermatitis, drug rash, and allergic contact dermatitis.

KEY MESSAGE:

• Potentiation of histaminergic itch by IL-33 depends on mast cells and IL-13 but not its activity on sensory neurons

• IL-33R expression is increased in lesional skin of patients with chronic spontaneous urticaria

ABBREVIATIONS

CSU

chronic spontaneous urticaria

His

Histamine

i.d.

intradermal

IL-33R

IL-33 receptor

ImmGen

Immunologic Genome Project

i.v.

intravenous

LM

littermate

NS

no significance

OSM

oncostatin M

OVA

ovalbumin

scRNA-seq

single cell RNA sequencing

WT

wild-type