Evaluation of Serum Zonulin Level and Intestinal Permeability in Patients with Chronic Idiopathic Urticaria and the Relationship between Serum Zonulin Level and Disease Severity

doi:10.21203/rs.3.rs-3342316/v1

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Evaluation of Serum Zonulin Level and Intestinal Permeability in Patients with Chronic Idiopathic Urticaria and the Relationship between Serum Zonulin Level and Disease Severity

https://doi.org/10.21203/rs.3.rs-3342316/v1

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Background

The development of Chronic Spontaneous Urticaria (CSU) and increased intestinal permeability may be related.

Aim

It was aimed to evaluate the role of intestinal permeability in the etiopathogenesis of CSU and the relationship between disease severity and zonulin levels by measuring the zonulin level, which is an indicator of the increase in intestinal permeability, in the serum of CSU and control groups.

Methods

61 CSU patients and 59 healthy controls were included. Demographic characteristics, personal and family history, urticaria activity score, age of onset, duration of last attack, antihistamine dose used, and concomitant diseases of CSU patients were recorded.

Result

Between the patient and control groups, there was a statistically significant difference in zonulin levels (p=0.000). Patients with angioedema had considerably greater zonulin levels than patients without angioedema, and there was a statistically significant difference in zonulin levels depending on whether angioedema was present (p=0.023).

Conslusion

These findings suggest that intestinal permeability may play an important role in the pathogenesis of CSU and angioedema.

chronic spontaneous urticaria

chronic idiopathic urticaria

autoimmune urticaria

anti-IgE

anti-FcεR1

intestinal permeability

zonulin

Chronic Spontaneous Urticaria (CSU) is a chronic in which etiopathogenesis has not been fully understood yet and which is thought to have autoimmunity in the etiology 45% of patients.
The relationship between CSU and autoimmunity diseases is remarkable, and there is an abnormal autoimmune response and state extending beyond the skin in CSU.

What does this study add?

This study reveals that increase in intestinal permeability may play an important role in the pathogenesis of CSU and angioedema.
In CSU patients, increase in intestinal permeability can be a milestone in elucidating the pathogenesis of the disease by explainin the abnormal autoimmunity, and can lead to new potential therapeutic targets and new treatment options.

Urticaria is a disease affecting the superficial dermis with erythematous, edematous, itchy papules and plaques. Since it lasts longer than 6 weeks, it is known that the disease becomes chronic.[1] While 80% of patients with chronic urticaria go into remission after 1 year, it has been shown that the disease still persists in 11% of patients after 5 years.[2]Although the etiopathogenesis of chronic urticaria is not known exactly, autoimmunity is held responsible in approximately 45%.[3] Although the exact mechanism of the pathogenesis of chronic urticaria is yet to be clarified, there are two primary theories that has been suggested. The first theory proposes that mast cells and basophils suffer from defects in their function or traffic due to dysregulation of intracellular signaling pathways, while the second theory suggests the development of autoantibodies against FcεRIα or IgE in these cells.[4]

Recent studies have revealed that impaired intestinal barrier function may initiate autoimmune diseases and exacerbate existing autoimmune diseases in individuals with genetic predisposition.[5]

Intestinal barrier function is regulated by physical, biochemical, and immunological mechanisms. The physical structure of this barrier consists of enterocytes, tight junctions (TJ), adherent junctions, desmosomes, mucus, and commensal bacteria.[6, 7]

When the intestinal barrier is disrupted, it can be referred to as the leaky gut. Increased permeability of the intestines leads to an uncontrolled entry of exogenous antigens, commensal bacteria, and bacterial products into the organism. In this situation, antigen traffic cannot be controlled and the submucosal innate immune system is stimulated. If this cycle persists, the acquired immune system is stimulated. Stimulation of the immune system leads to the release of cytokines such as IFN- γ, TNF-α, and more antigen passages. As a result, the inability to control antigen traffic enters a vicious cycle.[5]

In our study, we measured zonulin levels in serum to evaluate intestinal permeability.

Intestinal barrier control is provided by tight junctions (TJ) between enterocytes. Zonulin, on the other hand, is a protein that plays a role in the regulation of antigen traffic by controlling TJ.[8]

Zonula occludens toxin (Zot) was discovered during vaccine development studies against Vibrio cholerae bacteria.[8] Zot's discovery sheds light on the mechanisms involved in the regulation of paracellular transit in the gut. Zot is an endogenous enterotoxin that rapidly and reversibly regulates TJs.[9] It was thought to exert this effect by mimicking a protein regulating TJ and this protein was named Zonulin.[10] Proteomics analysis of human serum revealed that zonulin is pre-HP 2, the precursor of haptoglobin (HP). Pre-HP 2, defined as zonulin, has been shown to increase permeability in the jejunum and ileum in ex vivo studies. Zonulin also prevents microorganism colonization of the small intestine by increasing permeability. It also plays a role in the defense mechanism with this effect on natural immunity.[11]

Increased zonulin levels have been observed in autoimmune disorders like Type 1 Diabetes Mellitus, Multiple Sclerosis, Inflammatory Bowel Disease, and Celiac Disease, as well as metabolic disorders like insulin resistance, Type 2 Diabetes Mellitus, and Polycystic Ovarian Syndrome, as well as systemic infectious diseases like sepsis and HIV, and intestinal disorders like irritable bowel syndrome.[8]

To the best of our knowledge, there is no previous study evaluating intestinal permeability with serum zonulin levels in CSU disease. In this study, we aimed to evaluate the role of intestinal permeability in the etiopathogenesis of CSU and its relationship with disease severity by analyzing serum zonulin levels.

Study design and participants

This study was conducted over the period from April 2021 to December 2021 at the Dermatology Department in Dışkapı Yıldırım Beyazıt Hospital. Prior to their enrolment, all individuals provided written, informed consent. We recruited 122 participants, including 63 patients with CSU (30 males and 33 females; 18–62 years old with an average age of 32.96) and 59 age- and sex-matched healthy controls without CSU and other skin diseases.

As a result of the power analysis using the G*Power 3.1.9.2 program; A total of at least 120 samples was found to be sufficient, with an effect size of 80% power, 5% margin of error, d = 0.496.

All participants were Turkish and inhabitants of Ankara, Turkey. The diagnosis of KSÜ was based on clinical criteria. Patients who applied to the hospital and volunteered to participate in the study were included. Included in the study were patients who applied to the hospital and offered to take part. Patients with CSU who fulfilled any of the subsequent requirements were disqualified: age between 18 and 65; use of systemic antibiotherapy; obesity; co-occurring systemic inflammatory disease; autoimmunity; current infections; and history of gastrointestinal tract disease. The control group was made up of healthy individuals who were matched for sex and age and met the identical inclusion and exclusion requirements as CSU patients.

Measures

The age, weight, gender, marital status, self- and family history of CSU patients were recorded. When their disease started, how long their attacks lasted and the presence of angioedema accompanying their disease were questioned. Patients were classified as mild, moderate, and severe urticaria according to the urticaria activity score. The Weekly Itch Severity Score and the Weekly Hives Severity Score are two validated patient-reported outcomes for pruritus; the weekly score range is 0 to 21. Lower scores indicate less symptoms. UAS7 total scores range from 0 to 42. UAS7 score ≤ 6 is well controlled, 7–15 is mild, 16–27 is moderate, and

28–42 can be considered as severe urticaria.

Sample collection, biochemical and molecular analysis

All participants had their venous blood drawn, and the samples had to be brought to the biochemistry lab in no more than two hours. The serum samples were separated into separate Eppendorf tubes and kept at -80° until the day of analysis. Samples were centrifuged at 3000 rpm for 10 minutes. On the day of analysis, serum samples were removed according to the manufacturer's instructions and kept at room temperature (18–25°) for 30 minutes. In the analysis, Human Zonulin levels were measured in serum samples by "sandwich enzyme-linked immunosorbent assay" (Sandwich-ELISA, Elabscience®, Catalog Number: E-EL-H5560, Wuhan, China) kit.

The sensitivity to chitin was 0.47 ng/mL. The intra-assay coefficient of variation (intra-assay coefficient of variation, CV) ranged from 4.85–5.69%, while the inter-assay coefficient of variation (inter-assay, CV) ranged from 4.35–5.19%, per the manufacturer's data.

Statistical analysis

The Statistical Package for Social Sciences (SPSS), version 15.0, Chicago, Illinois, was used for all statistical analyses. Frequencies and percentages were used to show descriptive categorical data, and mean and standard deviation were used to display continuous data. When determining measurement values that fit the normal distribution, parametric approaches were applied. The measurement values of two independent groups were compared using the "IndependentSample-t" test (t-table value technique), in accordance with parametric approaches. The Kolmogorov-Smirnov test was developed to examine the distribution of continuous data for normality. For measurement data that did not meet the normal distribution, nonparametric techniques were applied. The measured values of two independent groups were compared using the "Mann-Whitney U" test (Z-table value) in accordance with nonparametric approaches. The association between two qualitative variables was examined using Pearson's 2 test statistics. Binary Logistic Regression was utilized to determine the variables influencing illness risk status using a backward LR model. Statistics were judged to be significant for P-values under 0.05.

1.The characteristics of participants

In this study, we enrolled 63 patients with CSU and 59 age- and sex-matched healthy controls. All of the subjects were of Turkish descent and originated in the same region. The patients' ages ranged from 18 to 62 years old, with a median age of 32.0. 52.4 percent of the patients were female. The average BMI was 25 kg/m2 (range, 17.9 to 29.8). Age, gender, and BMI did not significantly differ between the two groups. The characteristics of the study groups were shown in Table I.

A statistically significant difference in zonulin levels was discovered between the patient and control groups (p = 0.000), with the median zonulin level in the patient group being 49.3 [2.2–58.3] ng/mL and the median zonulin level in the control group being 41.9 [2.2–56.3] ng/mL.

Mild, moderate, and severe CSU patients classified according to UAS participated in our study. When we evaluated the patients according to severity, no statistically significant difference was found between serum zonulin levels and disease severity.

CSU patients were composed of male and female patients. When we evaluated serum zonulin levels according to gender, they were statistically significantly higher in women than in men.

Patients with angioedema had considerably greater zonulin levels than patients without angioedema, and there was a statistically significant difference in zonulin levels depending on whether angioedema was present (p = 0.023). Table II shows the correlation between zonulin levels and gender, disease severity, and angioedema presence.

In this study, we investigated whether intestinal permeability plays a role in the pathogenesis of CSU by looking at serum zonulin levels. In our study, we found statistically higher serum zonulin levels in CSU patients compared to the control group.

Autoimmunity, stress, pseudoallergens, dysbiosis, metabolic syndrome, and various drugs are held responsible as predisposing factors in the pathogenesis of CSU. Autoimmunity is a strong predisposing factor considering the presence of concomitant autoimmune diseases, positivity of various autoantibodies, higher incidence in women, and response to omalizumab, an IgE monoclonal antibody.

It is thought that the intestine plays a role in the formation of autoimmune diseases and that the intestine has a great influence on the initiation of the process.¹⁰ It has been shown that disruption of the integrity of the connection between enterocytes in the small intestine causes an increase in intestinal permeability, and the physiologic modulator of TJ between enterocytes is a protein called zonulin. [8, 10, 12]

The two main elements that trigger the release of zonulin are gliadin and bacteria.⁸ Many enteric pathogens affect the host intestinal tight junction, causing enterotoxin production. In addition to enteroxins, many enteric pathogens, including Escherichia coli and Salmonella typhi, cause intestinal zonulin release becomes.[11] When gliadin is applied only to the apical surface, both cell causes the release of zonulin through CXCR3 receptors in both culture models and intestinal tissue ex vivo studies.[8]

There is no previous study investigating intestinal permeability by looking at serum zonulin levels in CSU patients.

Zonulin increase has been associated with autoimmune diseases such as celiac disease, type 1 DM, inflammatory bowel diseases, and multiple sclerosis in recent years.[8] In these diseases, it is thought that the increase in intestinal permeability may occur together with and sometimes before the clinical picture of the disease and is also thought to be related with the severity of the disease.[10]

Drago et al. examined duodenal biopsies from celiac patients and controls ex vivo and showed that gliadin exposure caused a transient increase in intestinal permeability in both groups, more exaggerated and resistant in celiac patients, and that this increase was secondary to zonulin release.[13] The demonstration of increased zonulin in celiac disease has led to new treatment studies. Larazotide acetate, which blocks the zonulin receptor, is a molecule undergoing Phase III studies to be used in the treatment of celiac disease.[14]

In one study, when celiac patients were loaded with gluten and given larazotide acetate, a decrease in intestinal permeability, a decrease in symptoms such as diarrhea and bloating, and a decrease in anti-tissue transglutaminase antibodies and INF-γ levels were shown.¹⁵ In animal studies, it has been demonstrated that intestinal permeability increases prior to the onset of IBD and increases in first-degree relatives of IBD patients even when IBD is not present. [16, 17]

In our study, zonulin levels were higher in patients with CSU compared with the control group, suggesting that increased intestinal permeability may play an important role in the pathogenesis of CSU. However, serum zonulin levels were not associated with disease severity, which may be because there were not enough patients in each group according to UAS7.

In our study, zonulin levels were significantly higher in women than in men; there is no literature on the relationship between zonulin and gender. Zonulin levels were higher in women, suggesting that increased intestinal permeability in women may be one reason why autoimmune diseases are more common in women than in men.[18]

However, the fact that zonulin levels were higher in male CSU patients compared to the control group strongly supports the role of zonulin in the pathogenesis of CSU.

In our study, serum zonulin levels were significantly higher in CSU patients with angioedema compared to those without. Angioedema can lead to increased intestinal permeability by increasing extravasation of fluid in the intestines, increasing intestinal edema. The increase in intestinal permeability in the presence of angioedema suggested that zonulin may also cause an increase in vascular permeability. Because angioedema is also known to be an edema due to the permeability of the blood vessels. Although there is no publication in the literature investigating the effect of zonulin on blood vessels, there is a study showing that it regulates lymphatic endothelial cell layer structure and permeability.[19]

It has been shown in the literature that zonulin levels may increase before clinical manifestation.[17] Considering the results of our study, the idea that an increase in zonulin level in a patient with urticaria may be a harbinger of angioedema gains strength.

These findings suggest that intestinal permeability may play an important role in the pathogenesis of CSU and angioedema.

Consent for publication: This paper submitted with consent and knowledge of all co-authors.

Conflicts of interest: None

Funding: None

Patent consent: All patients supplied consent with the awareness that this information might be made publicly available, according to the consent for the publication of recognized patient data or other identifying material that was received by the authors and included at the time of article submission to the journal.

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Table I. Demographics and clinical caharacteristics of participants

Characteristic	CSU patients (n=63)		Healthy controls (n=59)		p
Male / Female (n)		30 / 33	29/ 30		0.865
Age (range)		34.95 ± 11.68 (18-62)	34.93 ± 9.07 (18-53)		0.743
BMI (Body Mass Index)		24.79 ± 3.03	24.28 ± 3.19		0.360
Cigarette pack year (+/-)		19/49	17/41		0.919
History of alcohol use(+/-)		5/58	3/56		0.525
Disease time (month)		47.95± 89,34 (2-396)	-
Last attact time (week)		8,91±12,67(0-72)
Plaque residence time		4.46±4.02(0-24)
Serum zonulin level (range)		45.99±10.71(2.2-58.3)	37.63±15.09(2.2-56.3)	*0.000

Table II. The relationship between zonulin levels gender, disease severity, presence of angioedema.

Characteristic		Serum Zonulin Level		P
Sex	Female(n)	33	51.1 (30.3-58.3)	*0.006
Sex	Male (n)	30	44.2 (2.2-56.6)	*0.006
Angioodema	(+)	22	51.6 (30.8-58.3)	*0.023
Angioodema	(-)	41	47.5 (2.2-56.6)	*0.023
UAS7	Well control	11	43.78 ±14.94	0.187
	Mild urticaria	22	43.64±12.12
	Severe activity urticaria	30	48.62±6.81

Note: Significant p values are marked in bold.

Mann-Whitney U C test for the comparison between study groups.*P<0,05

No competing interests reported.

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Evaluation of Serum Zonulin Level and Intestinal Permeability in Patients with Chronic Idiopathic Urticaria and the Relationship between Serum Zonulin Level and Disease Severity

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INTRODUCTION

MATERIALS AND METHODS

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RESULT

DISCUSSION

CONSLUSION

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References

Tables

Additional Declarations

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