Changes and imbalance of Th1 and Th2 immune response in pediatric patients with seasonal allergic conjunctivitis

1 Introduction

Ocular allergies are one of the most prevalent eye disorders seen in clinical practice. Allergic conjunctivitis (AC) is allergy-induced inflammation of the conjunctiva, the membrane covering the white part of the eye [1]. The European Academy of Allergy and Clinical Immunology classifies AC as ocular allergy and ocular non-allergic hypersensitivity and distinguishes two types of ocular surface hypersensitivity disorders. Seasonal AC (SAC) is the most prevalent form of AC, accounting for over 90% of the ocular allergies, and can be caused by immunoglobulin E (IgE)-mediated mechanisms [2]. Disease severity impacts the quality of life; visual impairment is observed in severe cases [1,3].

T lymphocytes activate and regulate the immune system through the cytokines they produce, which chemically signal other cells in the immune system, influencing their actions. Of all the cell types in the body, helper T (Th) cells are considered the most notable producers of cytokines. Among the two main Th cell types, Th1 cells work to eliminate invaders such as viruses and intracellular bacteria, while Th2 cells destroy pathogens such as bacteria and parasites outside the cells [4]. In addition to Th2-type cytokines that have a role in ocular allergic disease pathology, other inflammatory cytokines – like the typical Th1 cytokine interferon gamma (IFN-γ) – are also overexpressed during the active inflammatory phase of ocular allergic diseases [5]. IFN-γ released from Th1 cells during the inflammatory response is the most potent inducer of neopterin production, the levels of which may also be altered during allergic inflammation. IFN-γ also activates the indoleamine 2,3-dioxygenase (IDO) enzyme, which initiates the process of breaking down the essential amino acid tryptophan (Trp) through the kynurenine (Kyn) pathway [6].

The knowledge on Trp metabolic changes, immune system activation, and IgE levels in such patients remains limited; therefore, we sought to investigate their possible relationship with SAC. In this preliminary study, we measured (i) serum Trp and Kyn levels to calculate the Kyn/Trp ratio as an IDO activity indicator; (ii) neopterin levels as a Th1 response indicator; and (iii) IgE levels as a Th2 immune response indicator in pediatric patients with SAC. For this purpose, serum samples of 44 patients were assessed, along with those of 33 healthy pediatric participants, and correlations between the detected biomarkers and AC symptoms were investigated.

2 Materials and methods

3 Results

Table 1 displays the characteristics of the study groups, as well as the average symptom and sign scores in the patient group. The total symptom and sign scores were calculated for each patient, which indicated the severity of the allergy. The mean symptom and sign scores of the patients were 6.9 ± 2.9 and 7.2 ± 3.3, respectively.

Serum neopterin levels were examined as a biomarker of the Th1 immune response. As presented in Table 2, serum neopterin was significantly higher in patients with SAC (8.99 ± 0.81 nmol/L) than in healthy controls (6.42 ± 0.46 nmol/L) (p = 0.018), with a 1.40-fold difference.

Serum IgE levels were measured to evaluate the Th2 immune response (Table 2). The levels in patients with SAC (199.42 ± 2.41 ng/mL) were 1.97-fold higher than in healthy children (101.36 ± 0.97 ng/mL), and the difference was statistically significant (p < 0.001).

IDO activity was expressed as the Kyn/Trp ratio, calculated using the individual measurements of Trp and its main metabolite Kyn in serum samples (Table 2). Trp levels in patients with SAC were significantly lower than in healthy controls (64.97 ± 1.81 μM vs 72.15 ± 2.37 μM, p = 0.017). Kyn levels were also lower in patients with SAC than in healthy controls (2.77 ± 0.16 μM vs 3.13 ± 0.21 μM), albeit with no statistical significance (n.s.). The resulting Kyn/Trp ratios in patients with SAC and healthy controls were 46.49 ± 2.83 μmol/mmol and 41.05 ± 2.13 μmol/mmol, respectively, with no statistically significant difference (n.s.).

Correlations among the measured parameters in the patient group are presented in Table 3. Neopterin was significantly correlated with all serum and other parameters in the patient group, except for the sign score (Table 3). Among these, only the serum Trp levels were negatively correlated with neopterin levels (Rs = −0.334; p = 0.027). Furthermore, serum IgE and Trp levels in the patient group were negatively correlated (Rs = −0.302; p = 0.046). The Kyn/Trp ratio also showed significant correlations with Kyn (Rs = 0.732; p < 0.001) and Trp levels (Rs = −0.458; p = 0.003).

The correlations found in the healthy group were not consistent with those in the patient group. As presented in Table 4, only Kyn was correlated with neopterin (Rs = 0.356; p = 0.042) and the Kyn/Trp ratio (Rs = 0.819; p < 0.001).

4 Discussion

AC is an IgE- and mast cell-mediated inflammation of the conjunctiva, and SAC accounts for about 90% of all AC cases. Mast cell degranulation triggers the release of inflammatory mediators such as histamine, prostaglandins, cytokines, and chemokines. The signs and symptoms of AC can affect the patients’ quality of life and even pose a risk to their vision [9]. Research and clinical trials are growing as a result of the increased prevalence of allergy illnesses and their influence on health expenses and productivity [2]. Because AC affects many individuals, understanding its underlying mechanisms and discovering new, fast, and reliable biomarkers that would help clinicians tailor effective and novel treatment approaches are important.

Neopterin, a marker of cellular immune activation that can be readily detected in biological fluids using sensitive ELISA tests, has become essential in the diagnosis and monitoring of numerous diseases [8,10,11,12,13,14,15]. Neopterin is associated with immune responses in which Th1 cells are particularly active [16]. Although the Th2 response is at the forefront in allergic diseases, the Th1 response is also considered to contribute to and affect the severity of allergic reactions [17]. In the present study, neopterin levels were found to be significantly higher in the SAC group than in healthy pediatrics. Similarly, previous studies reported higher neopterin levels in patients with diseases underlying allergic conditions [18,19,20,21].

IgE-mediated allergy responses are the typical cause of AC. Histamine and other inflammatory chemicals are released during these reactions, causing inflammation and ocular discomfort [22]. A higher neopterin level in patients with SAC is plausible because the inflammatory process begins with the immune system response and may indicate the severity of inflammation in the disease course. Consistent with our findings, in recent studies [18,19], neopterin levels were found to be higher during urticaria attacks than during post-attack. Importantly, these studies did not detect any correlation between increased serum neopterin levels and disease severity, similar to our results showing no correlation between neopterin levels and symptom scores of the patients. A dysregulated immune response was also observed in children with allergic asthma who have increased neopterin levels compared with healthy controls [21]. Other studies [20,21] also found increased neopterin levels in atopic patients allergic to tree or grass pollen and allergic asthmatic children. Besides, neopterin levels did not correlate with the asthma control grade [21]. Considering the results of our own study along with these reports, high serum neopterin levels may suggest immune activation in SAC. During the seasonal period of the allergy, neopterin levels can be expected to increase as immune cells become active, serving as an indicator of the immune system’s response to allergens. Increased neopterin levels may also be a result of the immune system response becoming more complex in long-term and chronic AC. Based on our preliminary study results, larger studies evaluating the utility of neopterin level alterations in monitoring treatment response among patients with AC may be useful.

AC is a type I hypersensitivity immunological response of the conjunctiva, with IgE as the primary immune reactant [9]. Th2 cells generate cytokines that cause B cells to produce IgE. The released IgE binds to a receptor on mast cells, and the allergen cross-links, which leads to the release of inflammatory mediators [2]. SAC is an IgE-mediated ocular allergy problem. When an allergen enters the body and interacts with the immune system, hypersensitivity reactions lead to an increase in the IgE antibody [17]. Our study showed that higher IgE levels were associated with higher neopterin levels. Although the Th2 response is considered dominant in allergic diseases, the interaction of Th1 and Th2 responses may be more complex in some pathologies [17], as both IgE (Th2) and neopterin (Th1) are directly related to the immune system response. Thus, the pathophysiology of allergic disorders may be better understood by monitoring both IgE and neopterin levels. While serum neopterin and IgE levels were positively correlated in our patient groups, more clinical and mechanistic studies are needed to fully understand the relationship between IgE and neopterin [23,24]. Accordingly, in-depth studies should determine how changes in neopterin levels affect the IgE-mediated responses or how they alter the course of allergic diseases. Nevertheless, current data showing the association of neopterin and IgE with immune system disorders such as allergic diseases support the notion that combining these biomarkers would enable more precise clinical diagnoses.

Trp is an essential amino acid and a substrate for several proteins utilized by different cells in the human body [25,26]. Two distinct biosynthetic pathways are used to metabolize Trp: the production of Kyn derivatives and the neurotransmitter serotonin [27]. Kyn is the primary breakdown product of Trp, with IDO as the main enzyme in this degradation pathway [25,26]. IDO is preferentially and strongly induced by the Th1-type cytokine IFN-γ [26,27] and has been suggested to play a role in the pathogenesis of allergic diseases [24,27,28,29].

In our study, the Kyn/Trp ratio did not significantly differ between the patient and control groups but tended to be higher in patients, in parallel with neopterin and IgE levels. Meanwhile, Trp levels were significantly lower in patients with AC than in controls. Similar to our study, in patients with pollen-induced allergic rhinitis, Trp breakdown was found to be significantly increased during the pollen season [27]. In another study, there was a significant increase in IDO levels in atopic individuals with allergic rhinitis in comparison with controls without allergy. Also, IDO and total IgE levels were positively correlated. Despite the significant increase in IDO levels, age, sex, severity score, length of illness, nasal and blood eosinophilia, and number of positive allergens did not significantly correlate with IDO levels [30].

Despite the aforementioned findings, some studies reported opposing results. In a case–control study, suppressed IDO activity was shown in allergic disorders. Moreover, higher Trp and Kyn concentrations and a decreased Kyn/Trp ratio were found in atopic patients allergic to tree or grass pollen [20]. However, the significantly high neopterin levels in this study were in parallel with our findings. Another previous study reported significantly increased Trp and Kyn levels in pediatric patients with asthma, while the Kyn/Trp ratio was not changed. Additionally, none of the parameters were correlated with asthma control grade [21]. Significantly decreased IDO activity was also reported in the sputum and peripheral blood of allergic children [31]. Decreased IDO activity was found in patients with persistent food allergy. As in our study, Kyn levels were comparable between the patients and healthy controls, while Trp levels were higher in allergic patients [28].

The degradation and metabolism of Trp may play an important role in the pathophysiology of AC. Trp metabolism via the Kyn pathway may trigger inflammation and exacerbate allergic reactions. In addition, the utilization of Trp in serotonin production may worsen ocular symptoms by increasing inflammation and vascular permeability. These opposing mechanisms highlight the need for more research to identify the fundamental causes of AC. In this present study, while the significant decrease in Trp levels may reflect the Th1 immune response, indicating inflammation in allergic reactions, it may also be a result of the serotonin pathway, another breakdown pathway of Trp. Serotonin is a mediator that triggers inflammation and increases vascular permeability [32]. The conversion of Trp to serotonin may be important during allergic reactions [29]. Vascular dilation and fluid leakage in the eye may increase the symptoms of conjunctivitis [33]. In an earlier study, Trp degradation was correlated with the extent and severity of the disease [34]; however, in the present study, there was no correlation between Trp breakdown and the symptoms and signs of the illness, as with neopterin and IgE levels. Immune system cells may promote the metabolism of Trp via the Kyn pathway, which may increase inflammation and allergic symptoms. Likewise, the conversion of Trp to serotonin production may accelerate the inflammatory process in the eye. Apart from the pathophysiology of the disease, factors such as nutrition, genetics, or environment may affect Trp levels [20].

This study has some limitations. First, we did not measure other inflammatory markers or other Trp catabolism markers like serotonin. Second, we did not collect samples from the included patients outside the allergy season and thus were unable to compare these findings. Nevertheless, our findings support the complex involvement of both Th1 and Th2 cytokines in allergic diseases, suggesting the possibility of simultaneously identifying several biomarkers that may serve as surrogates for distinct disease pathways.

In conclusion, elevated neopterin levels in allergic diseases such as SAV may be considered an indicator of immune system activity and inflammation.