Response of FcεRI‐bearing leucocytes to omalizumab in chronic spontaneous urticaria

Abstract Background The pathogenesis of chronic spontaneous urticaria (CSU) and the mechanism of action of omalizumab in CSU remain unclear. Objective In this study, we assessed the responsiveness and FcεRI expression of various subsets of leucocytes in patients with CSU treated with omalizumab. Methods In this prospective cohort study, 30 patients were treated with 6 administrations of 300 mg omalizumab every 4 weeks, followed by a follow‐up period of 12 weeks. FcεRI expression and the percentage of basophils, monocytes, and dendritic cell subsets were analysed before and during treatment, and after follow‐up. In addition, anti‐IgE– and C5a‐induced basophil degranulation was measured. The results were correlated with disease activity and response to omalizumab. Results In addition to a rapid and significant reduction in FcεRI on basophils, we demonstrated a reduction in FcεRI on plasmacytoid dendritic cells during omalizumab treatment, which persisted until 3 months after discontinuation. FcεRI expression on basophils and its reduction did not correlate with the treatment response. Omalizumab led to an increased percentage of basophils in blood but not of the other FcεRI‐bearing leucocytes. Basophil responsiveness was differentially affected; anti‐IgE–, but not C5a‐induced basophil degranulation increased during the treatment. Apart from clinical non‐responders showing a stronger increase in anti‐IgE–induced basophil degranulation over a period time, no differences were found in omalizumab responders vs non‐responders. Conclusions/Clinical Relevance FcεRI expression on basophils decreased rapidly, while anti‐IgE–induced degranulation significantly increased due to omalizumab treatment in patients with CSU, persisting at least for 3 months after stopping the treatment. None of the markers were able to predict the effectiveness of treatment. Whether basophils play a role in omalizumab responsiveness in CSU remains unclear.


| INTRODUC TI ON
Chronic spontaneous urticaria (CSU) manifests as a skin disease with a sudden onset of weals, which last longer than 6 weeks. The disease duration ranges from 1 to 5 years or even longer in more severe cases. 1 Omalizumab is effective as a third-line treatment in a majority of CSU patients with insufficient response to a fourfold dose of antihistamines. 2,3 It is administered subcutaneously and reaches peak serum concentrations after an average of 7-8 days.
Clearance of the monoclonal antibody is slow, with a terminal half-life of 19-22 days, 4 which allows for relatively long treatment intervals of 4 weeks. A rapid clinical response can be seen in a proportion of the patients after the first omalizumab dose administration; however, other patients require multiple doses to reach a well-controlled disease status. 5 Omalizumab is a humanized monoclonal antibody, which binds to the Cε3 domain of free IgE, thereby preventing it from binding to Fc epsilon RI (FcεRI). 6 Depletion of free IgE by omalizumab leads to a down-regulation of the FcεRI on mast cells in a majority of patients. 7,8 Mast cells are considered to be the most important effector cells in CSU. In addition, a role for basophils has been suggested in certain urticaria phenotypes. 9 Basophil numbers are inversely related to urticaria severity. 10,11 An increased presence of basophils in the skin and decreased numbers in peripheral blood suggest that basophils are recruited to the affected skin sites. [12][13][14] Recent studies investigating the response of skin mast cells to omalizumab in allergic patients showed down-regulation of FcεRI expression after 1-2 months. [15][16][17] Therefore, other cell types, such as basophils and dendritic cells, might account for the rapid clinical effect of omalizumab. 18,19 Decreased degranulation of basophils after stimulation via FcεRI was demonstrated in patients with urticaria compared to that in healthy controls. 10 It is not known if responses to other stimuli, such as C5a (which activates basophils via a G-protein-coupled pathway), are affected. 20 Besides basophils and mast cells, other myeloid cells can also express FcεRI on their surfaces. 21 The presence of FcεRI has been demonstrated on monocytes and different types of dendritic cells, more profoundly in patients with allergies than in healthy individuals. In allergic rhinitis patients, expression of FcεRI on the different myeloid cells depended on serum IgE concentration, and treatment with omalizumab reduced the expression of FcεRI on basophils, mast cells, and DCs. [22][23][24] Recently, Deza et al suggested that the baseline expression of basophil FcεRI was a potential immunological predictor of responsiveness to omalizumab in urticaria. Furthermore, they found that patients who responded to omalizumab treatment had a rapid reduction in the levels of basophil FcεRI during treatment. Given the potential role of FcεRI-bearing leucocytes, in particular basophils, in the pathogenesis and/or treatment response to omalizumab, we evaluated the role of FcεRIbearing cells in CSU patients treated with omalizumab. Routine administration of immunosuppressants, including prednisolone and cyclosporine A (CsA) was discontinued with washout periods of 3 months prior to treatment with omalizumab. If prednisolone was used as a rescue medication, a washout period of 2 weeks was maintained.

| Design and population
After a screening period of up to 2 weeks, eligible patients received six doses of 300 mg omalizumab every 4 weeks. After the last omalizumab administration, the patients were observed during a follow-up period of 3 months. The patients were kept on a treatment with fourfold dose of H1 antihistamines throughout the study period.
As a rescue medication, patients were allowed to use prednisolone, up to 30 mg daily. All other CSU-related medications were discontinued.
Disease activity was measured throughout the study using the UAS7. 25 Treatment response is defined as a UAS ≤ 6 at week 24 of treatment. Improvement by a minimal important difference (MID) is defined as a reduction in 10 UAS7 points. 26 All the patients provided written informed consent, and the study was approved by the local ethics committee (protocol number 15-167). basophil degranulation, FcεRI, high-affinity IgE receptor, omalizumab, skin, therapy response, urticaria collected from nine self-reported healthy controls for baseline analysis. All serum samples were allowed to coagulate for 60 minutes. Serum and plasma were obtained by centrifugation and stored at −80°C. Total IgE was determined using the ImmunoCap assay according the manufacturer's instructions (Thermo Fisher Scientific).

| Leucocyte subset determination
Leucocytes subsets were identified using an antibody panel con-     with a median UAS7 score at baseline of 31.5 points were enrolled in F I G U R E 1 Median values of UAS7 for responders and nonresponders improve during omalizumab treatment. Median values of UAS7 at baseline, during omalizumab treatment, and during follow-up are presented for responders and non-responders. At the start of week 20, the final dose of omalizumab was administered, which initiated the follow-up period after week 24 (dotted line). Subjects who restarted omalizumab during the follow-up period were excluded from data analysis.

| Clinical efficacy of omalizumab
Subjects with UAS7 > 6 at week 24; n = 15 non-responders/partial responders; Subjects with UAS7 ≤ 6 at week 24; n = 15 responders; Overall median + confidence interval Week UAS7 score Follow-up the study. Patient characteristics (Table S1) corresponded with the CSU population in our clinic and current studies in literature. 27 Figure 1 shows the weekly median values of UAS7; the patients were differentiated into omalizumab responders and non-responders.
Fifteen patients (50%) showed a UAS7 score of six or lower (median 0) at 4 weeks after the last omalizumab administration (24 weeks) and were defined as responders. Fourteen patients showed a UAS7 score of seven or higher (median 16) at week 24 and were defined as non-responders. The UAS7 score of one patient was missing at week 24 and was marked as non-responder based on the last known UAS7 score.

| FcεRI expression on basophils, pDCs and mDC CD1cs decreases during treatment
In peripheral blood, we determined FcεRI expression on basophils, monocytes, pDCs, and two subsets of mDCs (mDC CD141 and mDC CD1c) at specific time-points before, during, and after treat- outlier with a total IgE > 5000 kU/L, a moderate correlation between baseline total IgE and baseline basophil FcεRI expression (r = .4, P = .037) was seen, which was comparable to that mentioned in a recent study. 28

| Only basophils percentages increase, other FcεRI-bearing leucocytes remain stable
We measured percentages of basophils, monocytes, pDCs, mDCs There was no significant change in median percentages of the other analysed leucocytes, such as eosinophils, monocytes, pDCs, mDC CDC141 + , and mDC CD1c + after omalizumab treatment (data not shown).

| D ISCUSS I ON
In this study, the effect of omalizumab treatment in CSU on different FcεRI-bearing leucocytes was investigated.
A decline of FcεRI on basophils was observed within 1 week after initiation and during omalizumab treatment, as shown in earlier studies. 7,8 In this study, we present several important and novel findings. The decreased FcεRI expression on basophils persisted up to 12 weeks after the last dose and was also detected on the mDC CD1c subset and pDCs. However, this decrease was not found on monocytes or on the mDC CDD141 subset. We also observed that the basophil responsiveness was differentially affected by omalizumab treatment, since anti-IgE-, but not C5a-induced basophil degranulation increased during the treatment. we found a large overlap in the FcεRI levels between healthy controls, responders, and non-responders, and no statistical difference was found among the three groups. Moreover, we did not find a statistical difference (P = .408), when complete responders (n = 9, median UAS7 = 0) were compared to extreme poor responders (n = 7, UAS > 16, median: 25). Moreover, the change in FcεRI levels did not correlate with the change in urticaria activity (UAS7 scores) per patient. A possible explanation for the discrepancy between the two study results might be a difference in patient population.
Although demographics between the two studies were fairly similar, patient-reported outcomes differed noticeably, since 81% of the patients in the study by Deza et al achieved a UAS7 score of ≤6 or a ≥90% reduction in UAS7 vs only 50% in our study.
The decreased FcεRI expression levels persisted in patients for at least 3 months even after discontinuing the omalizumab treatment. In a similar study, Jörg et al 30 found that FcεRI expression on basophils was decreased during omalizumab treatment and up to 2 months after the last dose. These results suggest lasting effects of omalizumab, which might explain why a proportion of the patients show a beneficial effect even after long intervals. 31 The median percentage of peripheral blood basophils showed a rapid and significant increase over time (Figure 3), which has been described previously during omalizumab and steroid treatment. 7,11,32 None of the other leucocytes showed such a reaction to omalizumab treatment, which may support a prominent role of ba-    be a reflection of the different maturation state of basophils due to decreased tissue inflammation, which in turn reduces the number of basophils in the skin and potentially leads to a lesser amount of basophil differentiation in the bone marrow.
Neither anti-IgE-nor C5a-induced basophil activation was related to treatment response. However, increase in anti-IgE-mediated basophil activation was most apparent in samples from patients not responding to omalizumab ( Figure S1). None of the other cellular responses showed a significant difference between responders and non-responders; therefore, we were not able to elucidate their role in the omalizumab treatment. These findings imply that the omalizumab-induced basophil changes might be responsible for the underlying clinical effects, but a yet unknown additional cellular effect could also play a role towards the favourable clinical response.
Notably, both the decrease in FcεRI expression on the cell surface of basophils, and the increase in anti-IgE-mediated basophil stimulation and decrease in C5a-mediated basophil stimulation continued for at least 3 months after discontinuation of omalizumab.
Given the relative short lifespan of basophils, this suggests either a prolonged effect of omalizumab or involvement of a more complex (possibly intracellular) mechanism of action of omalizumab.
Omalizumab induced a rapid and sustained decline of FcεRI expression on the surface of basophils, pDCs, and mDC CD1c. Despite the diminished expression of FcεRI on basophils by omalizumab, basophil degranulation was significantly increased after the cross-linking of FcεRI to anti-IgE. However, none of the findings could predict the response of omalizumab treatment, and more research is therefore required. Our findings suggest that response to omalizumab in CSU patients may be partly explained by pathways involving a high-affinity IgE receptor of the basophils.

ACK N OWLED G EM ENTS
We thank Stefan Nierkens for helping in the design of this study. We

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.