IgE and high‐affinity IgE receptor in chronic inducible urticaria, pathogenic, and management relevance

Abstract Background IgE and high‐affinity IgE receptor (FcεRI) expression on basophils have been scarcely explored in patients with chronic inducible urticaria (CIndU). Objectives To investigate baseline serum IgE and FcεRI expression on blood basophils in a large cohort of CIndU patients and its relationship to treatment response. Methods Baseline total serum IgE and basophil FcεRI expression measured by flow cytometry in 165 patients with CIndU was studied. The relationship of both parameters with the response to antihistamine and anti‐IgE (omalizumab) treatment was considered in a subsample of CIndU patients. FcεRI expression in basophils was assessed by mean fluorescence intensity (MFI) and basophil FcεRI standardized density (receptors/cell). Results The median FcεRI expression standardized per density in blood basophils was found significantly higher in patients with CIndU compared to HCs. A positive correlation was found between IgE serum levels and basophil FcεRI expression. Basal FcεRI expression was not related to antihistamine treatment response. However, it was related to omalizumab, and patients responding to omalizumab showed higher basal basophil expression of FcεRI levels. Non‐responders to the antihistamine showed significantly higher IgE serum levels. Conclusions FcεRI receptor overexpression in patients with CIndU shows almost the same pattern than chronic spontaneous urticaria. It seems to be independent of CIndU subtypes. Although additional studies would be welcome, our work highlights the relevance of FcεRI receptor regulation in CIndU supporting autoimmune basophil and mast cell activation and may be a biomarker for response to anti‐IgE therapy.


| INTRODUCTION
Among the two types of immunoglobulin E (IgE) receptors, 1,2 low (FcεRII) and high-affinity (FcεRI) IgE receptors, the latter is constitutively and primarily expressed on mast cells and basophils, where it binds the Fc region of IgE, an immunoglobulin isotype implicated in hypersensitivity and inflammatory and allergic processes. [3][4][5][6][7] Total IgE serum levels are usually slightly elevated in patients with chronic urticaria (CU) and has been described associated with different response to anti-IgE therapy. 8 In this regard, basophil FcεRI expression has already been characterized in chronic spontaneous urticaria (CSU) and it was assessed related to the treatment outcome with anti-histamine and anti-IgE therapy. [9][10][11] The classification of CU into spontaneous and inducible subtypes, and its further subdivision into other subtypes, 12,13 highlights the need to expand the field's knowledge of CU subtype specificity and differentiation. These phenotypic features of CU may influence therapeutic and treatment decisions, despite seemingly common clinical expression in some cases or overlapping features. 14 Therefore, a separate study is needed in CIndU as the phenotypic element may be affecting patient's characteristics and, feasibly, the role of IgE in the different diseases.
CIndU is a common inflammatory skin condition characterized by the recurrence of itchy wheals and/or angioedema lasting more than 6 weeks and induced by physical or environmental triggers. 12,15,16 CIndUs can occur alone or in combination with other types of CIndUs or CSU. The worldwide prevalence of CIndU is not negligible. Even pediatric population shows an estimated CIndU prevalence around 6%, 3% and some studies describe a 17% (26/153) of elderly CU patients that develop an active CIndU. 17 The prevalence of CIndUs has increased over time 18 and they have a considerable impact on quality of life. 19 Overall, the pathophysiology of CU involves activation and degranulation of effector cells, mainly skin mast cells, among others.
FcεRI expression is understood to play a key role on these cells in patients with CSU. 9 However, despite its putative importance in the pathogenesis of all CU forms, to date there are no studies focused on specifically characterizing the role of IgE serum and basophil FcεRI receptor expression in CIndU.
Thus, the main purpose of this preliminary study is to determine the role of IgE serum levels and FcεRI expression on basophils in a large sample of patients with pure or exclusive CIndU. Furthermore, taking into account the effect of omalizumab on FcεRI expression in CSU 9,20 and that clinical response to omalizumab could be predicted by baseline IgE levels in CSU 21 but also in CIndU, 22 in the present study we also assessed whether there are differences in the basal expression of basophil FcεRI receptor in patients with pure CIndU according to whether or not they responded to antihistamines or omalizumab. clinical history and the results of standardized provocation testing. 16 The classification of CIndU subtypes was also characterized. Part of this sample has already been included elsewhere. 11 As the main aim of the present study, peripheral blood samples from patients with CIndU were analyzed to measure basal total IgE in serum and FcεRI expression in basophils by flow cytometry. To avoid potential interferences, patients who were under treatment with biological therapies, oral corticosteroids, and/or other immunosuppressive agents were excluded from the study.

| Participants and study design
In addition, peripheral blood samples from a control group of 34 sex-equivalent healthy adult controls (HCs) with no family or personal history of allergic asthma, allergic rhinitis, CU and atopic dermatitis were included for reference data.
Complementary tests included in the study belong to the normal clinical practice in our daily medical activity. Ethical approval for the study was granted by the local Clinical Research Ethics Committee (ethics approval #2012/4913/I).

| Basophil cell preparation and flow cytometry for the high-affinity IgE receptor (FcεRI) expression
We followed standard procedures to perform flow cytometry analyses on both patients and HCs. 9 FcεRI expression in basophils was assessed by mean fluorescence intensity (MFI) and also by density Samples were lysed and fixed using FACS Lysing Solution (BD Biosciences) and analyzed by flow cytometry in a FACSCanto II using the FACSDiva software. At least 2 � 10 5 events were acquired.
Basophil FcεRI receptor levels were expressed as MFI. In addition, a standardized and more stable FceRI density measure (receptors/cell) was obtained since the MFI measure can be affected by different parameters. 23  To ensure consistency of analysis, the same investigator processed and analyzed all samples.

| IgE in serum and antithyroid antibodies (ATAs) levels
Total IgE and circulating antithyroid antibodies (ATAs) levels in serum were analyzed by a chemiluminescence immunoassay technique using the IMMULITE 2000 XPi System (Siemens).

| Urticaria control test (UCT) and provocation testing
The total score of the urticaria control test (UCT) 24  (EMO Systems GmbH). 27,28 With this, baseline provocation thresholds were evaluated.

| Treatment management
Treatments were applied following the EAACI/GA 2 LEN/EDF/WAO guideline for the management of urticaria. 25 The percentage of patients responding to antihistamine at licensed and fourfold up-dosing dose as well as to anti-IgE therapy, omalizumab, was evaluated. Patients who obtained after 4 weeks with antihistamine and 6 months with omalizumab an UCT ≥12 are considered responders if it was not the case patients were considered not responders. Baseline FcεRI expression and IgE serum levels were evaluated in both groups of patients according to their therapeutics response.

| Statistical analysis
All measures were baseline determinations. Descriptive statistics were performed for each variable, using median and range for quantitative variables, and absolute (n) and relative (%) frequencies for categorical variables. To compare quantitative and qualitative variables between patients with CIndU and HCs, the Mann-Whitney U test and chi-square test were used, respectively. Likewise, the Mann-Whitney U test was used to compare basal FcεRI expression and IgE levels attending to the response to treatments (anti-histamine therapy and omalizumab). Exploratorily, we also compared UCT and CSTT and CTT scores from the cold provocation test when exploring basal FcεRI expression and IgE levels according to treatment response, in order to clarify outcomes related to the treatment response/nonresponse groups.
Spearman's Rho (r s ) correlation was used to assess the association of FcεRI receptor expression with IgE serum levels in the whole CindU sample and by subgroups according to treatment response.
Complementary, exploratory, and post-hoc analyses were performed to compare the main variables between CIndU subtypes using Dunn's nonparametric multiple comparisons test.
The loss of sample size (N) for each variable is shown throughout the results. All analyses were performed with Prism 8.0 software (GraphPad) and a p < 0.05 was considered statistically significant.  Table 2).   Table 1.

Complementary analysis comparing FcεRI expression between
CIndU subtypes showed no significant differences between subgroups in either MFI values (p > 0.08 in all cases) or density values (p > 0.8 in all cases). In this vein, there were no significant differences in total IgE serum levels between CIndU subtypes (p > 0.3 in all cases; Table 2 and Figure 2).

| Correlation between basal basophil expression of the FcεRI and total IgE in patients with CIndU
For the entire CIndU sample, we observed a positive and significant correlation between total IgE levels and both our MFI measure (r s = 0.708, p < 0.0001) and density measure (r s = 0.708, p < 0.0001), indicating that higher basal basophil expression of FcεRI levels was associated with higher IgE serum levels in patients with CIndU ( Figure 3). These results remained significant when assessing CIndU subtypes (r s > 0.426, p < 0.023 in all cases).

| Antihistamine treatment
A total of 73.8% responded to antihistamine therapy while the remaining 26.2% did not respond (see Table 1).
There were no significant differences in baseline FcεRI expression between responders and non-responders to antihistamine

| Omalizumab
Evaluation of response to omalizumab of patients suffering from CIndUs showed that 89.5% responded to treatment and that the remaining 10.53% did not respond (see Table 1). About 20 cold ur-

| DISCUSSION
This is the first study evaluating basal FcεRI receptor characteristics in patients with CIndU. Our data showed that there were differences trending on significance between CIndU patients and HCs in FcεRI levels in basophils by MFI, which were highly significant according to receptor density results. This was independent of CIndU subtypes.

| High-affinity IgE receptor (FcεRI) basal expression
The results of this study in patients with CIndU are in line with those reported for CSU samples, with patients with CIndU presenting higher basal FcεRI receptor levels than HCs. 9 In a complementary study we have compared these results with those from a cohort of CSU patients (N = 79) and there were no differences in basal receptor levels between patients belonging to these two groups (MFI: p = 0.4118; receptor density: p = 0.0844).

F I G U R E 4
Total IgE serum levels between the responders and non-responders to H1 antihistamine therapy. For illustrative purposes the figure does not show the outliers. Nevertheless, the results remain significant after removing the outliers. *p < 0.05 GIMÉNEZ-ARNAU ET AL.

| Antihistamine treatment
Data on basal FcεRI receptor and IgE serum levels according to the treatment approach may provide more information in this regard. As it was suggested, in those who do not respond to antihistamine therapy, and who will therefore require treatment with omalizumab, we found higher total IgE serum levels versus responders to antiH1.
In addition, in some cases (chronic acquired cold urticaria) the provocation test found greater disease severity in non-responders, which does not happen when we compare patients who are responders and non-responders to omalizumab. However, there were no significant differences in baseline FcεRI expression between responders and non-responders to antihistamine therapy although both showed higher FcεRI expression than HCs. As such, the data on the FcεRI expression per se does not serve as a predictive biomarker of response to antihistamines, as shown in CSU, 11

| Omalizumab
For adults, there is good evidence that omalizumab is efficacious in CSU, with it having a notable effect on basophil FcεRI receptor density. [9][10][11]31 Clinical evidence in CIndU is not as extensive, and based on small samples, but it is increasing. [32][33][34] The current literature is slightly contradictory, with some articles postulating that omalizumab shows less efficacy in CIndU compared to CSU, but this has not yet been addressed systematically, 34 while others support the use of omalizumab in the treatment of patients with antihistamine treatment-resistant CIndU. 33  With regards to total IgE serum levels, although the median in non-responders to omalizumab is well above what is considered the threshold for allergic response (>400 IU/ml) and what appears to be far removed from those presented by the respondents to omalizumab (170 IU/ml), the fact that no differences were found between both subgroups could be due to the size of the samples, since we only had four patients as non-responders to omalizumab. As it was reported in CSU these patients with cold urticaria not responding to omalizumab showed also a baseline total IgE <40 UI/ ml. 11,21 In addition, and paradoxically, the medians are in the opposite direction to what has been previously seen with CSU, with CSU responders to omalizumab showing higher baseline IgE levels 9 and high total IgE in CSU representing a high chance of responding to omalizumab treatment. 30 Therefore, these preliminary results need to be tested in larger samples and are inconclusive regarding the role of baseline IgE serum levels in predicting response to omalizumab in CIndU.
In this vein, although sample sizes for CIndU subtypes do not allow definitive conclusions to be drawn here, exploration of differences by CIndU subtypes shows that, in general, none of the evaluated parameters are affected by urticaria subtype. Therefore, our data suggest that treatment approaches should be similar for different CIndU subtypes.
A final interesting nuance is that we found no differences between subjects who respond completely or partially to omalizumab in any of the evaluated parameters. Thus, it seems that the "true/ pure" non-responders should show an absence of UCT modification and present an idiosyncrasy that makes them show the receptor as a clear biomarker and in a more differential way, without establishing a grade for patients on a continuum according to response level.

| Limitations
The current study presents some limitations. Although the current sample size is quite large, this is the first study in this type of patients and it would be interesting to confirm these findings in other, even larger samples, in order to establish statistically powerful comparisons between CIndU subtypes. Therefore, additional multicenter studies are needed to corroborate the current findings.

| CONCLUSION
In summary, the data on basal basophil FcεRI receptor levels and their distribution according to treatment response (to antihistamine and omalizumab) show that patients with CIndU have similar behaviour to patients with CSU patients in terms of baseline total IgE levels, basophil FcεRI receptor and their response to anti-IgE treatment, without these parameters being affected by induced urticaria subtypes.