The role of innate immunity in the formation of various variants of the course of seasonal allergic rhinitis

Introduction

Seasonal allergic rhinitis (SAR) is rightfully attributed to the leaders of allergic pathology from the position of prevalence [1][2]. In order to control SAR, technologies for topical identification of causally significant allergens are being developed very intensively; treatment and prevention approaches are being improved. Success in solving these problems can be based only on the knowledge of the mechanisms of disease immunopathogenesis [3–5]. The involvement of specific immune reactions according to the Th2 variant in the formation of atopy is beyond doubt [6][7]. Meanwhile, more and more data are accumulated on the involvement of antigen-independent immune processes in the immunopathogenesis of allergic diseases, including SAR [8][9]. Currently, no one doubts the postulate about the importance of innate immunity in the implementation of specific immune processes, as well as about the effects of adaptive reactions on the potential of the innate immune response. It is known that the innate response to allergen proteases, including pollen, can induce cytokines of epithelial origin IL-33, IL-25, and thymic stromal lymphopoietin, which lead to activation of lymphocytes of the innate immune system ILC 2 in order to release cytokines of type 2 in an antigen-independent manner [10]. The conditions associated with the activation of mast cells and ILC 2 create the orientation of differentiation of macrophage cells in the M2 phenotype and ensure their participation in the maintenance of inflammation and the clinical manifestation of SAR [11][12]. The pathobiological role of the listed factors of the innate immune response in maintaining local allergic inflammation is being actively studied [13]. Meanwhile, data on changes in systemic immunogenesis formed in response to the progression of local type 2 inflammation have been little studied and are quite controversial. At the same time, the dynamics of these processes may be significant in the choice of a clinical manifestation scenario, the prognosis of the SAR course.

The aim of this particular study was to study the dynamics of the parameters of the innate immune response in patients with various SAR course variants.

Materials and methods

A comparative prospective empirical study to assess the immune system indicators depending on the severity of the clinical SAR course was conducted at the Department of Clinical Immunology and Allergology on the basis of the allergologist-immunologist office of the consultative diagnostic department and the Laboratory of Clinical Immunology and Allergology of RostSMU. The study involved patients with SAR, divided into subgroups with mild (34 people) and moderate (28 people) variants of the course, both men and women aged 18 to 50 years.

The study consisted of two control points. The initial examination happened during the period of pronounced clinical manifestation, during the dusting season of a causally significant allergen, while contacting an advisory polyclinic, before the appointment of therapy. The second examination was during remission lasting at least two months outside the pollination season. The study took place during the period from July 2017 to March 2019.

The inclusion criteria were age, a confirmed diagnosis of mild or moderate SAR with at least two years of disease experience, and proven sensitization to pollen allergens, without the use of the ACIT method to control the disease. Pregnant, lactating, patients with comorbid somatic pathology in the stage of subcompensation or decompensation, with a newly diagnosed diagnosis of SAR, in the presence of signs of acute infectious pathology, were excluded from the study. All the patients signed informed consent to participate in the study in accordance with the protocol approved by the Local Independent Ethics Committee of the Federal State Medical University of RostSMU (Protocol No. 18/13 dated December 13, 2013). The diagnosis of SAR was made in accordance with Federal Clinical Guidelines for the Diagnosis and Treatment of Allergic rhinitis [14].

The anamnestic data, the results of clinical examination and the severity of the main SAR symptoms during the period of exacerbation were evaluated: nasal congestion, rhinorrhea, itching in the nose, and sneezing. In order to objectify the results of the clinical examination, the total nasal symptom score (TNSS) scale was used [15], as well as a standardized questionnaire on the quality of life of allergic rhinitis patients – Rhinitis Quality of Life Questionnaire (RQLQ) [16].

The methods of investigation during the dusting season and the clinical manifestation of SAR consisted in conducting and evaluating microscopy data of nasal mucus. Microscopy was performed after smear staining using the Romanovsky–Giemza method to characterize the cellular composition of mucus. Outside of the flowering season, in the absence of intercurrent acute respiratory viral infections, patients underwent scarification skin tests with water-salt extracts of pollen allergens. The degree of sensitization was assessed by the severity of the local reaction on the palmar surface of the forearm 20 minutes after sampling. Accounting was carried out in the following points: 0 – the size and appearance of the local reaction are similar to the reaction in the control; 1 – a blister of 2–3 mm in size at the scarification site, visible only when the skin is stretched; 2 – a blister of no more than 5 mm, surrounded by hyperemia at the scarification site, clearly visible when the skin is stretched; 3 – a blister of 6–10 mm, surrounded by hyperemia; 4 – a blister of more than 10 mm.

When characterizing the parameters of innate immunity in the peripheral circulation, the number of natural killers (NKs) (CD3-CD16+), the number of their cytolytically active forms containing intracellular granzyme B granules (CD3-CD16+GrB+), and the number of monocytes carrying TLR2 (CD14+CD282+) and TLR4 (CD14+CD284+) receptors for pattern recognition were estimated. For this purpose, flow cytofluorometry was conducted using Cytomics FC 500 (Beckman Coulter, USA) and sets of monoclonal antibodies with two and three-color labels corresponding to the tasks (manufactured by Beckman Coulter, USA). The formation of reactive oxygen species (ROS) by neutrophils was recorded using the NBT test, while evaluating spontaneous values (NBT sv), the results obtained under conditions of additional stimulation of neutrophils in vitro (NBT st) and calculating the coefficient of stimulation Kst. (NBT st./NBT sv.). Quantitative assessment of the content of cytokines in blood serum TNF-α, IL-6, IL-4, and IFN-γ was carried out by enzyme immunoassay on Vector-Best test systems (RF).

As a control group, the corresponding indicators of the immune system of practically healthy blood donors with no indication of a history of somatic pathology, including allergies, were evaluated only once.

Statistical data processing was carried out using the software package Statistica for Windows 6.1 (StatSoft, USA). In the first stage, descriptive statistics were carried out using exploratory analysis; the nature of the distribution of quantitative data was evaluated according to the Shapiro-Wilk criterion (since the sample sizes were >50 people). In cases where the sample obeyed the normal distribution law, the averaged data were presented in the form of mathematical expectation (M) and standard deviation (SD) M±SD. If the sample did not obey the normal probability law, then the median (Me) and the 25^th and 75^th percentile (Q1:Q3) were used in the description, presenting the data in the form of Me [Q1;Q3]. After that, a pairwise comparative analysis of independent data was performed, comparing alternately a group with a mild severity of the current/control group, a group with a moderate severity of the current/control group, a group with a mild degree/a group with a moderate severity. If the distribution of data (for each factor) was subject to the normal probability law in both groups being compared, a parametric Student's t-test was used to check the significance level of the differences. If, however, in at least one of the two groups compared, the factor data were not distributed according to the normal probability law, and also when both samples did not obey the normal probability law, the nonparametric Mann-Whitney criterion was applied. The differences between the groups were considered statistically significant at p<0.05; in the opposite case, the differences were considered statistically insignificant (p>0.05).

Results

The data on the clinical characteristics of patients allowed distinguishing two groups of subjects: group I (34 people) – with a mild degree of severity of the course of SAR, and group II – (28 people) with a medium-heavy version of the course. The patients were comparable in age (30±5.5 and 33±5.7 years, respectively), gender (men – 41 and 46; women – 59 and 54), the length of illness (7.7±2.7 and 10.8±3.5 years), the duration of the acute SAR period (2.9±1.7 and 3.6±1.9 months), and the presence of hereditary predisposition according to the results of allergic anamnesis (47% and 46%).

Rhinorrhea, burning and tickling sensations in the nose, difficulty in nasal breathing, itching, and paroxysmal sneezing were noted in all patients with a mild variant of the clinical SAR course. The data from the analysis of the tables of the main symptoms showed that nasal congestion was the most significant, and the score reflecting this sign was less pronounced with immediate assessment (i-TNSS 1.59±0.5 in the evening, 1.59±0.5 in the morning) than with retrospective assessment (r-TNSS — 1.97±0.30 in the evening and 1.94±0.34 in the morning). The sum of the i-TNSS scores (1.29±0.14) was also slightly lower than the total score of the r-TNSS scale, which was 1.44±0.21. The average value of the total score of the RQLQ quality of life assessment questionnaire in a subgroup of patients was 80.6±8.86, which is almost two times less than the maximum possible when using this test. It should also be noted that the highest average value is the indicator reflecting the inconvenience caused by the general symptoms of the disease (17.2±3.45), and the lowest (7.1±1.82) – by sleep disorders.

The analysis of the indicators of the innate link of the immune response demonstrates an increase in the production of ROS by neutrophil granulocytes. This fact is confirmed by an increased indicator of the spontaneous NBT test in comparison with the control parameters (97.0 [ 90.0; 107.0] CU, in the control 90 [ 88; 91], p=0.012). At the same time, with additional stimulation of neutrophils in vitro, oxygen-producing activity was lower (150.0 [ 142.0; 171.0] cu) than in healthy (203 [ 192;217], p=0.011). This fact is reflected in the reduced value of the NBT-stimulation coefficient (1.60 [ 1.50; 1.70], in the control 2.0 [ 1.8; 2.1], p=0.011). The nature of changes in the parameters of the lymphoid component of innate immune protection demonstrated the preservation of the quantitative parameters of NKs: the relative number of circulating CD16+ lymphocytes did not differ from the data of the control group (respectively 10.50 [ 7.00; 13.00] and 12 [ 10.00; 13.00], p=0.86). At the same time, one should note the inhibition of the functional properties of these cells, which is more noticeably confirmed by a decrease in the absolute amount of NKs containing lytic granules of granzyme B (0.084 [ 0.058;0.124] 109/l, in the control 0.14 [ 0.11; 0.16], p=0.011). The data reflecting the ability of peripheral blood monocytes to recognize pathogen-associated patterns did not differ from the control values of the number of CD14⁺CD282⁺ (59.5[ 49.0; 70.0] and 64 [ 55.0; 70.0], p=0.15) and CD14⁺CD284⁺ cells (20.0 [ 11.0; 30.0]% and 20 [ 15.0; 23.0], p=0.078). It should also be noted that there are no statistically significant differences in serum levels of immune response mediators.

As for the patients of the second subgroup, which consisted of people with a moderate variant of the SAR course, the indicators of objectification of TNSS symptoms varied around the maximum level (3 points). The “nasal congestion” criterion was most pronounced, which was noted both with retrospective (r-TNSS 2.96±0.19 in the evening and 2.93± 0.26 in the morning) and immediate assessment (i-TNSS 2.96±0.19 in the evening and 3.00± 0.00 in the morning). The factor of the total RQLQ score also looks very clear: its average value in the subgroup was only 15% less than the maximum possible, amounting to 143.2±5.28. At the same time, most of the points were determined by the “general symptoms” factor (34.8±3.87).

While characterizing the parameters of cellular factors of the innate defense system, there were no changes in the receptor expression of peripheral blood monocytes in relation to practically healthy ones. So, the relative number of CD14⁺CD282⁺ (60.50 [ 50.50; 68.00]%) and CD14⁺CD284⁺ mononuclears (20.00  [ 12.05; 29.50]%) did not differ from the control (64 [ 55.0; 70.0]% at p=0.68 and 20 [ 15.0; 23.0]% at p=0.87, respectively); the number of circulating NKs and the spontaneous metabolic activity of neutrophils were also within the values of practically healthy donors. At the same time, the functional potential of these cell lines was reduced. This fact documents a decrease in the value of the stimulated NBT test (136.0 [ 127.0; 163.5] c.u., in the control 203 [ 192;217] c.u., p=0.011), as well as the coefficient of stimulation of NBT (1.50 [ 1.40;1.60], in the control 2.0 [ 1.8;2.1], p =0.019). In addition, there was a decrease in the relative number of circulating cytolytically active NKs (CD16⁺Gr⁺, % 6.00 [ 4.00; 10.00], in the control 10 [ 8; 12], p=0.02). The analysis of the cytokine spectrum of blood serum indicates shifts in this immune response system: the content of proinflammatory mediators IL-6 was significantly increased: 7.3 [ 5.1;9.4] pg/ml, in the control 2.0[ 1.2;2.4], p=0.01 and TNF-α [ 2.3 [ 1.7;3.4] pg/ml, in the control 1.6[ 1.4;1.9], p=0.01. In addition, the values of immunoregulatory oppositional cytokines IFN-γ (8.9 [ 7.0;11.2] pg/ml, in the control 6.2[ 4.9;6.8], p=0.02) and IL were increased (3.2 [ 1.9;4.3], pg/ml, in the control 2.1[ 1.0;2.4], p=0.03).

While solving the problem of searching for distinctive signs in the reaction of systemic indicators of innate immunity, depending on the variant of the course of SAR, a comparison of data characterizing a mild degree of clinical course with a moderate variant of SAR in the stage of exacerbation of allergic inflammation was carried out. A comparison of the clinical picture, based on a comparison of the scores of evaluation scales, revealed statistically significant differences in the i-TNSS criteria reflecting each of the symptoms, their average values of both morning and evening indicators, as well as the overall score: in the mild SAR case, its value was 1.29±0.14, and in the moderate case – 2.70±0.16, p<0.001. Similar dynamics can be traced when comparing the results of r-TNSS in values that characterize not only each of the symptoms, but also the total score of the average values of morning and evening indicators (1.44±0.21 for the mild variant, 2.65±0.27 for the moderate, p<0.001, respectively). The comparison of the quality of life assessment data also looks convincing. Thus, with a mild clinical course, the values of the RQLQ criteria ranged from 30 to 60% of the total score, and with a moderate variant, the spread of values was no more than 10% with a total increase to 85–90%.

A comparison of the indicators of the cellular link of innate immunity in patients with mild and moderate forms of SAR revealed no differences in the production of ROS by neutrophil phagocytes, in the expression of Toll-like receptors 2 and 4 by peripheral blood monocytes, in the number of circulating NKs and cytolytically active granzyme-positive CD16⁺ lymphocytes. At the same time, in patients with a moderate form, in comparison with patients with a mild variant of the clinical course, an increase in the serum content of all cytokines analyzed in the work is recorded. To a greater extent, this fact distinguishes the pro-inflammatory mediator IL-6 (Table 1).

It is also known that there is a persistent inflammation of the nasal mucosa in patients with SAR even in the absence of a causally significant allergen [17][18]. This circumstance determined the interest in analyzing the indicators of the innate immune response in the same patients, but in remission. The authors of this study found that with a mild variant of the SAR course, in comparison with the control parameters of practically healthy donors, the production of ROS by neutrophils in the in vitro additional stimulation test was reduced (NBT st., c.u. 141.5[ 125.0; 156.0] c.u., in the control 203[ 192;217], p=0.012). At the same time, the indicators of the spontaneous NBT test do not differ between the groups (respectively 93.0[ 80.0; 98.0] c.u. and 90 [ 88; 91], c.u., p =0.89). The attention is also drawn to the value of the parameter reflecting the functional properties of the lymphoid subpopulation of innate immunity. The number of CD16⁺Gr⁺ cells in the circulation was reduced (5.0[ 4.0; 8.0%, in the control 10 [ 8; 12], p=0.01). At the same time, there were no statistically significant differences in the expression of TLR 2 and TLR 4 by monocyte cells, and there were no differences in the serum content of immune response mediators. During the period of remission of the moderate SAR variant, in comparison with the control group, the parameters of the stimulated NBT test were reduced (136.0 [ 127.0; 163.5] c.u., p=0.02), a decrease in the proportion of functionally active CD16⁺Gr⁺ lymphocytes was recorded (7.30 [ 3.50; 9.50]%, p=0.014) while preservation in comparison with the values of control of quantitative indicators of NKs, expression by cells of the TLR2 and TLR4 monocyte series, the content of cytokines IL-4; IL-6; IFN-γ; TNF-α in the blood serum. A comparison of the data reflecting the functional potencies of the cellular components of innate immunity in the conditions of remission of moderate and mild SAR revealed no statistically significant differences in the metabolic activity of neutrophils, monocytic expression of TLR 2 and TLR4, lytic properties of NKs, and cytokine profile of blood serum.

Discussion

The stage of exacerbation of mild SAR manifests an increase in the metabolic activity of the neutrophil link and weakening of patients’ adaptive resources in relation to the parameters of practically healthy people. A decrease in functional potencies also distinguishes the population of NKs. In patients with moderate-severity SAR in the stage of exacerbation, in comparison with the control figures, changes in the functioning of the immune system are manifested by a decrease in the reserves of metabolic activity of neutrophil phagocytes and lytic potencies of NKs. A very significant factor is the systemic activation of cytokine production of both pro-inflammatory and regulatory orientation, while the content of IL-6 in the peripheral serum is increased to a greater extent, and the fact of increased synthesis of mediators of both Th1 and Th2 profiles is also very interesting. It should be noted that it is this circumstance – the production of immune response mediators – that distinguishes the systemic response of the innate immune response depending on the severity of the clinical SAR course. A very important result of the observation is the detection of changes in the functioning of innate immune response cells in patients with SAR compared with control criteria in the absence of causally significant allergens. Changes in mild SAR patients during clinical remission are determined by a decrease in the effector potencies of neutrophils and NKs. In the absence of pollination and clinical manifestations of SAR of the moderate course variant, the suppression of adaptive reserves of oxygen-dependent metabolism of neutrophil granulocytes and the cytolytic potential of NK cells continues to be recorded, and there are no fundamental differences from the data of the same period in patients with a mild course variant. If the peculiarities of the functioning of the immune system in patients with acute SAR are justified by the current allergic inflammation, then the quantitative and functional indicators of innate immunity that differ from the control data in the stage of clinical remission are objective evidence of a violation of the systemic immune response even in the absence of a causally significant allergen and at the earliest stages of the disease.

Conclusion

The totality of the presented data allows stating that changes in the response of cellular components of the innate immune response in SAR patients are formed in the earliest stages of clinical manifestation, are stable outside the season of pollination and severity, and the key manifestation of progression is cytokine imbalance. Inhibition of the adaptive potential of neutrophil phagocytes and the lytic activity of NKs can play a significant role in the development of complications of SAR caused by secondary immune insufficiency.