**1.3 Patient-reported outcome measures in Chronic Urticaria**

Patient-reported outcome measures are instruments of objective and subjective evaluation for the management of CU and are essential tools for assessing treatment effects in clinical trials.

As described above, CU-Q2oL and AE-QoL are the two questionnaires available for evaluating the CU burden on QoL. Instead, the Urticaria Control Test (UCT) is a valid and reliable tool to assess disease control in patients with CU and a score of ≥12 indicates well-controlled urticaria [1]. However, the most frequently utilized tool in clinical trials is the 7 days Urticaria Activity Score (UAS7) [1, 11]. It is also suitable for evaluation of disease activity by urticaria patients and their treating physicians. The UAS7 is based on the patient self-assessment of the two main urticaria signs and symptoms recorded once a day for 7 consecutive days:


The sum of score is 0–6 for each day, and 0–42 for the UAS7 (0 = urticaria-free; 1–6 = well-controlled urticaria; 7–15 = mild activity; 16–27 = moderate activity; 28– 42 = severe activity), respectively. Overall disease activity is best measured by advising patients to document 24-hour self-evaluation scores for several days.

For the patients affected by recurrent angioedema, alone or in addition to wheals, the last EAACI/GA<sup>2</sup> LEN/EDF/WAO guideline also suggests to use the Angioedema Activity Score (AAS) [1]. It consists of five items regarding the characteristics of angioedema to have occurred in previous 24 hours [11]. A score between 0 and 3 is assigned to every answer field. The question scores are added up to produce a daily score (0–15). Daily AAS can be summed to give 7-day (0–105), 4-week (0–420), and 12-week scores (0–1260) [12].

### **1.4 The Chronic Urticaria treatment guidelines**

As first-line symptomatic treatment for urticaria, the EAACI/GA<sup>2</sup> LEN/EDF/ WAO guideline suggests regular administration of second-generation, nonsedating, nonimpairing H1-receptor antihistamines due to their efficacy and good safety profile [1]. This class of drugs has a greater receptor specificity, lower penetration of the blood–brain barrier, and less likely to cause drowsiness or psychomotor impairment in comparison to the first-generation antihistamines.

In non-responders adult or paediatric patients, the second-line treatment is the up-dosing of the antihistamine by as much as 4-fold. For patients (aged 12 years and older) who have not responded to four-times the standard dose of secondgeneration H1-receptor antihistamine, omalizumab, a humanised monoclonal anti-IgE antibody, as add-on therapy is considered the third-line treatment. If there is no response to the omalizumab within 6 months, or if the condition is intolerable, the fourth-line treatment is the prescription of cyclosporine A (CsA), which inhibits the production of IL-2, IL-3, IL-4, and TNF-α in lymphocytes and the IgE-mediated release of histamine from mast cells. High doses of CsA and long duration treatment are associated with adverse events such as abdominal pain, nausea, vomiting, paresthesia, headache, hirsutism, elevated serum creatinine, and hypertension;

**Figure 2.**

*Simplified stepwise algorithm for the treatment of urticaria adapted from the current EAACI/GA2 LEN/EDF/ WAO guideline.*

however, these side effects resolve after reducing dosage [13]. Nevertheless, CsA should be avoided in patients with chronic kidney disease or poorly controlled hypertension. CsA at the dose of 3–5 mg/kg/day has been shown in small, doubleblind, randomised controlled trials to be effective in patients with CSU who do not adequately respond to antihistamines [14, 15]. During CsA treatment, given the significant side effects, the blood pressure, renal function, and serum CsA levels should be monitored regularly.

A simplified stepwise algorithm for the treatment of CSU adapted from the EAACI/GA<sup>2</sup> LEN/EDF/WAO guideline is summarised in **Figure 2**. At any moment, short courses of corticosteroids (e.g. prednisone 25 mg/daily) are admitted if symptoms are exacerbated or poorly controlled [1].

#### **1.5 The anthystamines limit in Chronic Spontaneous Urticaria**

In the pre-omalizumab period, treating CSU patients was a real challenge for physicians due to the low rates of response to H1-antihistamines, which were the only approved medication and the mainstay of symptomatic treatment. Two metaanalysis including studies published between January 1990 and November 2014 revealed that 63.2% and 38.6% of patients remain symptomatic despite treatment with licensed dose and updosed H1-antihistamines, respectively [16]. Another study reported even lower response rates to standard dosage, with disease control in only 22% of patients [17].

### **2. The role of anti-IgE antibodies in Urticaria**

The immune system is a network of cells, tissues, and organs that work together to defend the body against attacks by foreign invaders such as bacteria, viruses,

parasites, and fungi [18]. The host uses both innate and adaptive mechanisms to detect and eliminate pathogenic microbes, and both of these mechanisms include self-nonself discrimination. Immunoglobulins (Ig), also known as antibodies, are glycoproteins produced by white blood cells that are specific for an antigen (e.g., bacteria, viruses, parasites, or fungi), aiding its destruction by a cascade of downstream pathways. There are five primary classes of Igs (IgG, IgM, IgA, IgD and IgE), which differ in their biological features, structure, target specificity, and distribution [19]. Among them, IgE are involved in allergic reactions with a type I autoimmune mechanism.

### **2.1 IgE**

It is believed that IgE have evolved to protect humans from helminth infections, which are one of the major threats to human life. IgE molecules exist in a monomeric form consisting of two heavy and two light chains and are the most important participants in an allergic reaction [20]. When a foreign substance, called allergen, enters our body, a person with an inherited predisposition to this substance will begin to develop a specific type of IgE, which will evoke a cascade of reactions aimed to eliminating this allergen. IgE are present in serum at very low concentration (50–200 ng/mL in a normal individual) and have a very short half-life (1–2 days). However, tissue-resident IgE may persist for several days (approximate half-life of 2 weeks in the skin) [21, 22]. This may be due to the extremely high affinity of IgE for the IgE Fc receptor (FcεRI) and in particular its slow dissociation from this receptor, resulting in re-binding of the dissociated IgE to its receptors, and restricted diffusion away from the tissue within which it resides [23].

There are two structurally and functionally distinct receptors that bind with the Fc epsilon (Fcε) region of IgE: the high affinity FcεRI and the lower affinity CD23 FcεRII [24, 25]. Through their Fc portions, IgE molecules bind to the Fc receptors present on the surface of mast cells and basophils. The cross-linking of such membrane-bound IgE antibodies by multivalent antigens triggers the release of chemically active substances, such as histamine, leukotrienes, prostaglandins, and chemotactic factors, from the cells. These substances initiate allergic and inflammatory reactions and serve as a chemoattractant for other cells [26].

#### **2.2 Anti-IgE antibodies as Chronic Urticaria treatment: why?**

This is the first question claimed by the scientific community, since IgE are involved in allergic reactions and CU is not known as an allergic reaction. Before answering this question, we should know the mechanism of action of omalizumab in CSU. Omalizumab has been effective in the treatment of urticaria, believed to have an autoimmune origin, and in cases where the etiology is unknown [27].

There are several hypotheses regarding the mechanism of action of omalizumab in CSU patients. One of them is based on the fact that the density of IgE receptors on the surface of mast cells and basophils is proportional to individual patient's plasma IgE levels [28, 29]. It is hypothesized that omalizumab, by lowering free IgE levels in the blood and subsequently in the skin, may lead to down-regulation of a large percentage of surface IgE receptors, thereby decreasing downstream signaling via the FcεRI receptor pathway [30, 31]. Cell activation would then be diminished, and subsequent inflammatory processes, as complement activation and cellular infiltration, would be suppressed as well. As a consequence, the frequency and severity of symptoms of CSU would be lessened [28, 30, 31].

Another hypothesis is that omalizumab reduces the levels of circulating IgE, leading to a rapid and non-specific desensitization of cutaneous mast cells [32].

Subsequent effects, such as down-regulation of IgE receptor, may help to sustain the response. Serrano-Candelas et al. demonstrated comparable actions of omalizumab on mast cells and basophils while investigating the in vitro mechanism of action of omalizumab on these cells [33].

In a review by Kaplan et al. new insights into the potential mechanisms of action contributing to the efficacy of omalizumab in CIndU/CSU have been suggested based on both clinical and in vitro studies [30]:


Deza et al. investigated the effect of omalizumab on the basophil expression of FcεRI receptor in a cohort of patients with active CSU [34]. Patients exhibiting significant clinical improvement showed a sharp reduction in the levels of basophil FcεRI after 4 weeks (*p* < 0.0001), which was maintained throughout the total duration of the treatment.

In a study by Asero et al., omalizumab responders showed a dramatic decrease of D-dimer plasma levels after the first administration of the drug (p = 0.003), suggesting a possible effect of omalizumab on coagulation activation and fibrin degradation [32].

However, none of these theories fully account for the pattern of symptom improvement seen with omalizumab therapy. Therefore, additional research is warranted to further explain the involvement of omalizumab in relieving symptoms associated with the complex, multifactorial pathogenesis of CIndU/CSU.

#### **2.3 The Chronic Spontaneous Urticaria main endotypes**

CSU is a mast cell-driven disease. The initial event in the development of skin changes, such as sensory nerve stimulation, vasodilation and extravasation, as well as the recruitment of basophils, eosinophils, and T cells, which lead to whealing, itch, and angioedema is attributed to the degranulation of skin mast cells.

Two groups of mast cell degranulation signals have been so far identified and characterized in CSU pathogenesis: IgE autoantibodies to autoallergens and IgG autoantibodies that target activating mast cell receptors [30]. Therefore, it is now clear that there are at least 2 distinct pathways, type I and type IIb autoimmunity, that contribute to the pathogenesis of this complex disease [35]. In type I hypersensitivity to self, also called autoallergy, antigens crosslink the IgE on mast cells

and basophils to cause release of vasoactive mediators, while in type IIb hypersensitivity antibodies, usually IgG, bind to antigen on a target cell.

About twenty years ago, the demonstration of IgE autoantibodies against the thyroid microsomal antigen thyroperoxidase in the serum of a CSU patient, identified a possible role of type I autoimmunity in the pathogenesis of urticaria [36]. Many studies have further characterized the prevalence and pathogenic relevance of type I autoimmunity in CSU. In particular, CSU patients were found to express more than 2-fold higher IgE-anti-thyroperoxidase serum levels as compared to healthy control subjects (p < 0.001) [37].

Kolkhir et al. systematically evaluated the literature on the prevalence of thyroid autoimmunity in CSU and vice-versa, finding a positive correlation between CSU and elevated levels of IgG antithyroid autoantibodies with the studies reporting rates consisted in 10% [38]. Levels of IgG against thyroid peroxidase resulted more often elevated in CSU than those of other IgG antithyroid autoantibodies (strong evidence). Moreover, CSU patients exhibited significantly higher levels of IgG antithyroid autoantibodies (strong evidence) and IgE anti-thyroperoxidase (weak evidence) than controls.

However, IgE autoantibodies directed to a large assortment of autoantigens beyond thyroperoxidase are expressed in the skin of CSU patients as thyroglobulin, tissue factor, and interleukin (IL)-24 [39, 40]. Hatada et al. found that the antidsDNA IgE levels were significantly higher in patients with CU than in normal subjects, while no differences in the anti-dsDNA IgG levels were observed [41]. Furthermore, most of the studies confirm that IgE autoantibodies should be responsible for the increased total IgE levels in CSU patients in which, differently to the control subjects, most of the IgE was found to be directed against autoantibodies.

A type IIb hypersensitivity mechanism in which IgG autoantibodies against IgE were involved, was first described in CSU in 1988 [42]. Few years later, IgG autoantibodies directed to FcεRI, the high-affinity receptor for IgE on masthocytes and basophils, were also identified [42]. Grattan et al. introduced the Autologous Serum Skin Test (ASST) in CSU patients, consisting in eliciting with an intradermal injection of their own serum a wheal and flare response [43]. A positive reaction in the ASST confirm the presence of these autoantibodies.

CSU driving by type IIb autoimmune mechanisms is further supported by the basophil activation test [44]. The serum of a subpopulation of CSU patients stimulates heterologous basophils and this activity is due to the presence of autoantibodies against FcεRI as well as in positive ASST responses.

The two endotypes play a key role in inducing different phenotipe of the same disease: type I (autoreactive) and type IIb (autoimmune) CSU patients differ in some features, laboratory markers, and rates and speed of response to treatment [45]. In particular, type IIb autoimmune CSU patients have been suggested to have higher disease activity and longer disease duration as well as higher rates of autoimmune comorbidity. Basopenia and eosinopenia may also be more common.

A higher proportion of patients receiving omalizumab 300 mg achieved response as early as week 4 (early responders) when compared with placebo [46]. This is in line with type I autoimmune/autoreactive mechanism: anti-IgE rapidly binds free IgE, including IgE against autoantigens, and IgE/anti-IgE complexes bind autoallergens preventing mast cell degranulation. CSU patients that take more than a month (late responders) to respond to omalizumab, probably underwent a type IIb autoimmunity, where the reduction of free IgE results in the slow loss of membrane-bound FcεRI from skin mast cells [46].

New endotypes of CSU have been proposed in addiction by recent reports, suggesting a key role of the coagulation pathway factors, ligands of the Mas-related G protein–coupled receptor X2, basophils, and other signals in the pathogenesis of CSU [47, 48]. Moreover, other research to characterize better the role and the relevance of type I and type IIb autoimmunity in CSU and to support the existence of distinct and separate endotypes, are still in progress.

In contrast to CSU, autoimmunity in CIndU has not yet been described.
