Introduction

Chronic spontaneous urticaria (CSU) is a common disease in the community associated with a high disease burden on patients and the health system, including prolonged illness, frequent emergency and outpatient visits, sleep difficulties, and detrimental effects on work performance and social interactions.¹ Autoimmunity mediated by pathogenic autoantibodies is involved in the pathogenesis of the disease in about half of the cases, but the underlying pathomechanism could not be determined in the others.²

Omalizumab effectively improves symptoms in a significant proportion of patients with CSU, but its mechanism of action in CSU has not been fully elucidated.^3,4 Our experiences and evidence from previous studies have shown that in the significant proportion of patients who responded to omalizumab, it is unlikely to discontinue treatment for several years.^5,6

There are observations that patients with CSU often experience unusual emotional stress before disease onset and report symptoms that are worsened by stress. It has also been reported that many patients with CSU exhibit a variety of psychiatric disorders, most commonly anxiety, depression and perceived stress.¹ A recent meta--analysis of 25 studies revealed that almost one out of three patients with CSU has at least one underlying psychiatric disorder.⁷ The authors suggested that a ‘brain-skin’ link with local neuro-immune endocrine circuits may underlie the pathogenesis of stress-induced or exuberated allergic and inflammatory skin diseases.^7,8 Many observations on the effects of stress on the neuroendocrine and immune systems in psoriasis, atopic dermatitis and prurigo nodularis support this theory.^9–11 In this model, it has been suggested that emotional stress activates a hierarchical cascade of events which includes various neuronal mediators, such as nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and substance P (SP).^8,12,13

The skin is highly innervated by numerous nerve fibre subpopulations originating from the dorsal root ganglia, where neuropeptides are produced and stored. Mast cells are located perivascular close to the peptidergic C fibres in the upper dermis, an ideal location for sensory nerve stimulation.¹² Emotional stress and various external stimuli result in the depolarising of sensory nerve fibres, and subsequently release numerous mediators, including neuropeptides, neurotrophins and hormones stored in the cytoplasmic vesicles of nociceptive neurons from the axon terminals to the stimulated area antidromically.^12,13 This mechanism allows a rapid response to occur long before the immune system is stimulated.¹³ Neuropeptides released by stimulation of sensory fibres lead to mast cell degranulation. In turn, itch-related mediators including histamine, tryptase and NGF released from mast cells, also stimulate peptidergic C fibres, which store proinflammatory neuropeptides such as CGRP and SP. This bidirectional positive feedback between mast cells and sensory fibres exacerbates mast cell-mediated neurogenic inflammation and pruritus.^8,12,13

Skin mast cells are considered the primary effector cells in urticaria. In addition to IgE-dependent degranulation, there is evidence that many signals can target skin mast cells through IgE-independent mechanisms by various receptors, including G-protein coupled and complement receptors.^4,14,15 It was reported that emotional stress may cause urticaria symptoms by increasing mast cell degranulation via G-protein coupled receptors in a neuropeptide-dependent manner.^14,15

The aim of our cross-sectional, case-controlled study was to assess the role of psychiatric disorders in CSU, and the effect of omalizumab on NGF, CGRP and SP levels in patients who responded to omalizumab, apart from its anti-IgE effect. We also investigated stressful life events that occurred before the onset of illness in CSU patients.

Materials and Methods

Study population

This cross-sectional, case-controlled study was performed between August 2021 and November 2021 at the Department of Dermatology, Gazi University Faculty of Medicine Research Hospital which is a tertiary hospital in Ankara, Turkey. Patients who had been followed up in our specialised urticaria outpatient clinic and had received at least 6 months of omalizumab (300 mg/month) for CSU were included in the study.

In the patient group, age, gender, concomitant angioedema, concurrent chronic inducible urticaria, duration of CSU, duration of omalizumab, 7-day urticaria activity score (UAS7) and urticaria control test (UCT) scores were recorded.

We excluded individuals with any previous inflammatory or allergic skin disease history, a diagnosed psychiatric disorder, and the use of psychotropic substances within 6 months, marked cognitive impairment, or who received glucocorticoid therapy for at least 1 month. All participants provided informed consent. The study was approved by the Institutional Ethics Committee (Approval number: 07.2020.599).

Mental assessment and psychometric instruments

The levels of depression, anxiety and stress were assessed with the Depression Anxiety Stress Scale-42 (DASS-42), which is a self-administered questionnaire designed to measure the magnitude of three negative emotional states.¹⁶ DASS is a 42-item questionnaire consisting of three subscales, each containing 14 items scored on a four-point scale ranging from 0 (did not apply to me at all) to 3 (applied to me very much), measuring the extent to which each item was experienced over the previous week. The DASS depression focuses on reports of low mood, motivation and self-esteem; DASS-anxiety on physiological arousal, perceived panic and fear; and DASS-stress on tension and irritability.

Stressful life events were assessed with the Life Events Checklist for DSM-5 (LEC-5), a self-report measure developed to screen for lifetime traumatic events.¹⁷ The instrument assesses exposure to 16 different adverse life events that may potentially result in post-traumatic stress disorder or distress and includes one additional item considering any other extraordinarily stressful event not captured in the first 16 items.

Measurement of NGF, CGRP and SP levels

In addition to routine examinations, 5 mL of blood was taken from the patient and control group into the coagulation activator tube and centrifuged at 3000 rpm for 10 min at room temperature. In the next step, the serum was separated, transferred to the Eppendorf tube, isolated and stored at -80°C until the test time. After all sera were collected, serum NGF, CGRP and SP levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology Inc., 14780 Memorial Drive, Suite 216, Houston, Texas 77079 USA) according to manufacturer’s instructions.

Statistical analysis

Statistical analysis was performed using SPSS 27.0 (SPSS Inc., Chicago, USA). Baseline characteristics of cases and controls were analysed using descriptive statistics. We assessed the comparisons between groups for the categorical variables using the Chi-square test. The normality of continuous data was assessed by the Kolmogrov–Smirnov test. A comparison of the various parameters between patients and controls was performed by the independent sample t-test for parametric data. The Mann–Whitney U-test was used for paired group comparison of numerical variables when the variables were not normally distributed. Correlation analysis was performed using the Spearman correlation test. The data were described as mean (standard deviation [SD]) or median (interquartile range [IQR]), as appropriate. The level of statistical significance was set at p <0.05.

Results

A total of 42 CSU patients and 42 healthy controls (as a control group) were included in this cross-sectional study during the 4 months of enrollment. The demographic features and disease characteristics of the study groups are summarised in Table 1. Of 42 patients with CSU, 29 (69%) had concomitant angioedema and 26 (62%) had concurrent inducable urticaria. The median duration of omalizumab treatment was 21.5 months (IQR, 6–65). The median UAS7 and UCT scores were 6 (IQR, 2–15) and 17 (IQR, 16–18), respectively.

Among the 42 patients, 26 (62%) noted at least one stressful life event preceding the exacerbation of CSU, and 32 (74%) reported that their urticaria symptoms were exacerbated by stress. The median duration between the event and CSU onset was 3.5 months (range, 0 and 4 months).

There were no significant differences in all three variables in the DASS subscales between the patient and control groups (depression: p = 0.465; anxiety: p = 0.730; stress: p = 0.784) (Figure 1).

Figure 1 Comparison of depression, anxiety and stress levels between CSU patients and normal controls. p values were obtained by the independent sample t-test. DASS, Depression Anxiety Stress Scale; SD, standard deviation; CSU, chronic spontaneous urticaria; NC, normal healthy controls.

Serum NGF levels were significantly lower in the CSU group compared with the controls (median [interquartile range], 90.15 pg/mL [61.85–127.45], p <0.001), whereas CGRP levels were found to be significantly higher (47.30 pg/mL [23.82–111.22], p <0.001). There was no significant difference in SP levels between the two groups (Figure 2).

Figure 2 Comparison of NGF, CGRP and SP levels between CSU patients and normal controls. p values were obtained by the Mann–Whitney U-test. NGF, nerve growth factor; CGRP, calcitonin gene-related peptide; substance P, SP; IQR, interquartile range; CSU, chronic spontaneous urticaria; NC, normal healthy controls.

We also analysed the relationship between UCT and UAS7 scores and DASS42 subscales scores and levels of three neuromediators. Correlation analyses revealed that in CSU patients, UAS7 scores showed a significant positive correlation with DASS42 scale results for ‘anxiety’ (r = 0.448; p = 0.003) and a trend towards ‘depression’ (r = 0.285; p = 0.068). Moreover, UCT scores showed a significant negative correlation with the DASS42 scale results for ‘anxiety’ (r = -0.399; p = 0.009) and a trend towards ‘depression’ (r = -0.277; p = 0.076). Stress scores did not correlate with UAS7 or UCT scores (p>0.05). It was revealed that CGRP levels among neuropeptides showed a positive correlation with UAS7 scores (r = 0.326; p = 0.035) and a negative correlation with UCT scores (r = -0.331; p = 0.032). No correlation was shown for substance P and NGF (Table 2).

Discussion

In this study, we found that CSU patients who achieved symptomatic relief with omalizumab treatment had similar depression, anxiety and stress levels as that of healthy controls. These findings contradict the studies reporting a higher incidence of psychiatric comorbidities in patients with CSU,⁷ but support some recent studies showing that omalizumab treatment improves psychiatric parameters in patients with CSU.^18–20 In their study, Diluvio et al. revealed that CSU patients demonstrated markedly reduced psychological health subscale scores compared with controls. They showed that after 6 months of treatment with omalizumab, 71% of subjects who had obtained a borderline or abnormal score for depression received a normal score and 60% of subjects with a previous abnormal anxiety score achieved a normal score for anxiety.¹⁹ Similarly, Patella et al. observed that all the psychological scores (PSS for perceived stress, HADS-A for anxiety and HADS-D for depression) were improved after 24 weeks of treatment with omalizumab in refractory CSU patients.²⁰ A recent study with asthma patients has also shown that omalizumab reduced symptoms of depression and stress in these patients as well.²¹

In this study, almost two-third (62%) of CSU patients were found to have experienced a stressful life event a median of three and a half months before the onset of urticaria. These results concord with previous studies, which showed that patients with CSU had significantly more life events in the 6 months before the disease onset.^22–24

A recent meta-analysis reported no relationship between psychiatric illness and the severity of CSU,⁷ however, we found anxiety scores positively correlated with UAS7 scores and negatively correlated with UCT scores in patients with CSU. Moreover, there was a similar trend for depression, although it was not statistically significant. Our findings align with a study by Tat et al., who reported a significant positive correlation between UAS and anxiety and depression scores in patients with chronic urticaria.²⁵

In their recent systematic review, Konstantinou et al.²⁶ considered that stress-induced imbalance or irregularity of the neuro-immune-cutaneous circuit may contribute to the pathophysiology of chronic urticaria. Emotional stress increases neurogenic inflammation by both modulating the hypothalamic-pituitary-adrenal axis in the brain and skin and increasing neuromediator levels. As a result, many chronic inflammatory skin diseases may emerge and exacerbate.^12–14 A number of studies have shown that in numerous inflammatory and autoimmune diseases, including urticaria, tissue and serum NGF levels increase compared with healthy controls.^14,27,28

We found significantly lower levels of serum NGF in patients with CSU compared to controls. Bonini et al., in their study to compare serum NGF levels between patients with asthma and/or rhinoconjunctivitis and/or urticaria--angioedema and healthy controls, observed that serum NGF levels were higher in allergic diseases. They also showed a positive correlation between NGF levels and total IgE levels.²⁹ In another study in which mouse and human in vitro cultured mast cells were activated by IgE, NGF secretion increased as measured by ELISA and RT-PCR was shown.³⁰ Considering that omalizumab binds to free IgE in mast cells and inhibits IgE-mediated FcɛRI activation, these data reinforce that NGF release was decreased secondary to inhibition of IgE-mediated mast cell activation.

Nerve growth factor is an essential neuromediator for the survival of nociceptive neurons during development and neurogenic inflammation. Sensory nerve fibres in the skin end by branching towards areas with high NGF concentrations. Therefore, NGF determines the sensitivity threshold in the interactions between mast cells and nerve fibres, while a hyperreactive state occurs in pathophysiological conditions, resulting in increased skin sensitivity in the local environment to neurogenic flare, itch and pain.¹⁴ Taken together, omalizumab may increase the threshold of sensitivity of sensory nerve fibres to stress and other external stimuli by decreasing NGF levels.

Nerve growth factor also exacerbates neurogenic inflammation by causing increased expression of TRPV1 in nerve fibres, followed by the release of SP and CGRP, thus creating a vicious cytokine ‘pro-itch’ cycle.¹³ Joachim et al. showed that both stress exposure and injection of NGF up-regulated the percentage of SP+ or CGRP+ sensory neurons in murine skin.⁸ Substance P and CGRP are classical neuropeptides that mediate vascular effects by acting directly on vascular endothelial cells and smooth muscle cells. CGRP is one of the most potent microvascular vasodilator mediators known to contribute to most neurogenic vasodilation. Substance P increases vascular permeability with subsequent plasma extravasation and oedema.¹³ Some evidence has recently been presented that especially SP and CGRP neuropeptides may play a vital role in the pathophysiology of urticaria.^14,26 In a series of studies by Kimata et al.,^31–33 it has been shown that the stress of playing video games, listening to cell phones and exposure to road traffic increases allergen-induced wheal responses in parallel with increased plasma levels of SP, VIP and NGF. Borici-Mazi et al., in their study to compare the cutaneous responses to SP and CGRP in patients with chronic urticaria with healthy adults, observed the skin of patients with chronic urticaria increased sensitivity to the neuropeptide SP and CGRP.³⁴ In another study, lesional skin in CSU was found to contain significantly more CGRP than non-lesional skin.³⁵

We found that serum CGRP levels were markedly higher in the patient group compared with healthy controls, whereas there was no difference in SP levels. In parallel with our findings, Tedeschi et al.³⁶ reported that SP levels were not found to be higher in CSU patients than in controls, whereas several studies showed that circulating SP levels in CSU patients were higher than in healthy controls.^37–39 Despite several studies evaluating CGRP levels and their effects on the skin, little is known about circulating CGRP levels in patients with CSU. In a study conducted by Başak et al., in which they compared the neuropeptide levels of patients with chronic urticaria and healthy controls, it was found that no difference was found between CGRP levels while SP levels were significantly higher in patients with chronic urticaria compared to control.³⁹ Another study by the same author reported that antihistamine treatment did not affect serum SP levels but increased CGRP levels in chronic urticaria.⁴⁰ We obtained similar results in our study involving patients under omalizumab treatment. It has been reported that neuropeptides activate skin mast cells via G-protein-coupled receptors in a neuropeptide-dependent manner, not via FcɛRI receptors in an IgE-dependent manner.^14,15 These findings indicate that omalizumab may affect the levels of neuropeptides which are closely related to stress, apart from the decrease in free IgE and FcɛRI levels on mast and basophil cells, which is the known mechanism of action in urticaria.

Limitations

The limitations of our study include that since our study was a cross-sectional study, the depression, anxiety and stress levels of the patients before omalizumab could not be shown. Another limitation is that serum neuromediator levels had yet to be measured before omalizumab, as the literature, which suggested neuropeptides induce mast cells in an IgE-independent manner^14,15 was taken into account in our study. However, our study findings suggested that omalizumab may affect neuronal mediator levels as well as prevents IgE-mediated mast cell activation. Investigating the effects of omalizumab on neuropeptide and neurotrophin levels in CSU might also be another interesting subject for further studies.

Conclusions

We found that the depression, anxiety and stress levels of patients with CSU who benefited from omalizumab and received treatment for more than 6 months were similar to those of the healthy subjects. However, the fact that two-third of the patients reported a stressful life event before the disease suggests that stress may play a role in the onset and exacerbation of urticaria in suspected individuals. However, the fact that two-third of the patients reported a stressful life event before the disease suggests that stress may play a key role in the onset and exacerbation of urticaria in suspected individuals. The known action of omalizumab, reducing free IgE levels and down--regulating FcɛRI receptors, does not explain all of its effects in CSU. The striking differences in CSU patients treated with omalizumab for NGF and CGRP in our study compared to healthy controls suggest that omalizumab affects neuromediator levels associated with stress and neurogenic inflammation. Further prospective studies are needed to better understand the effect of omalizumab on psychiatric comorbidities and stress-related neuromediators in CSU and to confirm our results.