Introduction
The course of SARS-CoV-2 infection, which led to the outbreak of the COIVD-19 pandemic, varies greatly – from asymptomatic infection to a severe course, often ending in death [1]. We no longer have any doubts about the important role of immunological processes in the course of infections, which is best seen in the most severe ones [2]. Most studies to date have focused on this stage of the disease. Understanding the mechanism of severe infection gives an opportunity to develop methods of early diagnosis and treatment, and thus reduce the number of victims of the pandemic. But mild and asymptomatic infections are just as important to keeping the pandemic going. They are largely responsible for the rapid spread of the virus, despite attempts to contain it through social distancing and other non-specific methods [3]. The situation has also changed with the introduction of vaccination and the dominance of new variants of the virus, which have led to a reduction in the number of the most severe cases. Mild and asymptomatic patients predominate; therefore, it is important to better understand the mechanisms, including immunological ones, of these stages of the disease.
SARS-CoV-2 virus infection can be divided into three main phases: the viral infection phase, the pulmonary phase, and the systemic hyperinflammation phase [4]. Previous research has revealed the key role of the immune system, especially in the last phase, when its excessive, uncontrolled stimulation leads to damage to many organs. The phenomenon of overstimulation, expressed by the production of many cytokines, is called cytokine storm and is quite well known and described in the literature [5]. These studies led to the formulation of specific recommendations for diagnosis (the prognostic role of IL-6 concentration) [6] and treatment (the use of tocilizumab in severe cases) [7] of COVID-19.
Much less is known about the role of the immune system in mild and asymptomatic infections. But even in these patients, symptoms of immune system stimulation were found.
Inflammatory bowel diseases (IBDs), which include Crohn’s disease (CD) and ulcerative colitis (UC), are a group of autoimmune diseases, and therefore the role of the immune system is also significant in their pathogenesis. In the initial phase of the pandemic, many concerns were expressed about the safety of this group of patients, especially due to their widespread use of immunosuppressive drugs. However, these hypotheses were not confirmed, and studies in this group of patients allowed for a better understanding of the immunological mechanisms during SARS-CoV-2 infection [8–10].
Aim
The aim of this study is to assess the stimulation of the immune system expressed through the production of cytokines in IBD patients with asymptomatic COVID-19.
Material and methods
This is a multicentre prospective study evaluating serum cytokine levels in IBD patients who have had asymptomatic SARS-CoV-2 infection. Three tertiary centres (from Warsaw, Lodz, and Poznan) recruited participants for the study from 1 May to 30 September 2020, from patients who came to the centres for the next dose of a biological drug or due to an exacerbation of IBD. During the recruitment period, regulations limiting the movement of people were in force in Poland, and access to outpatient care as well as to planned hospitalisations was limited. Patients with symptoms of respiratory tract infection could not enter the centres – such people were sent home or directed to hospital wards treating COVID-19. For this reason, none of the patients in the study had symptomatic COVID-19.
Patient characteristics
The study involved 473 patients who made 1180 visits to the centres during the study. During each visit, patients filled in a form that was obligatory for the whole country, which assessed the occurrence of symptoms of respiratory tract infections in the last 7 days. In addition, information on IBD was collected, both the history of the disease and current activity. On this basis, demographic data (age, sex), type of disease, duration, history of surgeries, current treatment, and disease activity expressed by partial Mayo score (for UC) and Crohn’s Disease Activity Index (CDAI) (for CD) were assessed.
Laboratory analysis
At each visit, the patient had serum collected, which was divided into 2 samples. In the first one, the concentration of SARS-CoV-2 antibodies in the IgA + M and IgG classes was determined. SARS-CoV-2 IgG and IgM+IgA antibodies were measured by using the ELISA method, targeting viral spike (S) and nucleocapsid (N) antigens (Vircell Microbiologists®, Granada, Spain). Positive cut-off values were established according to the manufacturer’s instructions. The value expressed was the antibody index, which was defined as sample optical density/cut-off serum mean optical density × 10. It was 8 for antibodies in the IgA + M class and 6 for antibodies in the IgG class. All tests were performed in the Coronavirus Laboratory Diagnostic Unit of the Central Clinical Hospital of the Ministry of the Interior and Administration in Warsaw.
The second sample was frozen at –80°C immediately after collection for possible further testing.
All patients with positive IgA + M (n = 50) and IgG (n = 46) antibodies were included in the analysis. Eighteen patients had a positive result in 2 classes. Forty-one antibody-negative patients randomly selected from the remaining patients were also included in the analysis. In these patients, a panel of cytokines was tested from the second serum sample.
The concentration of the following cytokines was determined in these samples: interleukin (IL) 1a (IL-1a), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL- 15, IL-17, IL-23, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and TNF-β (measured by using the ELISA method, Quansys Biosciences®). All tests were performed in the Diagnostic Laboratory Unit of the Central Clinical Hospital of Ministry of Internal Affairs in Warsaw. According to the manufacturer’s recommendations, samples with a concentration below the detection limit were considered negative. Detection of a given cytokine at any concentration marked a positive result.
Statistical analysis
The analysis was conducted in statistical software R, ver. 4.2.1, with α = 0.05. The χ2 test and Fisher’s exact test were used to analyse whether the presence of cytokines was related to selected variables. The level of quantitative variables was compared between 2 groups using Mann-Whitney’s U test. Univariate logistic regression models were built, in which the presence of selected cytokines was considered as dependent variables. Multivariate models were built by a stepwise method. Initial models included variables that had a p-value lower than 0.25 [11] in the univariate models. Odds ratios were calculated with 95% confidence intervals. Nagelkerke’s Pseudo R2 and χ2 tests were calculated to evaluate multivariate models.
Results
Of the 119 patients, 58.8% were male, the mean age was 30 years. Of these, 75.6% had CD and 24.4% had UC. Most patients were in remission of IBD: median CDAI was 119.78, and median Mayo score was 3.00. Most were treated with 5-ASA (89.1%). There was a high percentage of patients treated with thiopurines (62.2%), as well as biologically: anti-TNF drugs (56.3%) and vedolizumab (29.4%); 19.3% were treated with steroids. Smoking was declared by 16% of patients.
Of all the cytokines tested, only in the case of IL-2, IL-6, IL-8, IL-10, and TNF-α were sufficient positive samples obtained to qualify these results for further statistical analysis.
Patients’ characteristics and antibody and cytokine results are presented in Table I.
Table I
Group characteristics
IL-2
In the group of patients with the presence of SARS-C0V-2 antibodies in the IgA + M class, a significantly higher percentage of people with an increased presence of IL-2 was found (52.3% vs. 29.6%; p = 0.021). This relationship did not apply to IgG antibodies (35.4% vs. 42.6%; p = 0.539). There were also significantly more smokers in this group (24.6% vs. 5.6%; p = 0.010). Analysing the use of drugs, a significantly higher percentage of patients taking adalimumab was found. However, the univariate and multivariate regression analysis showed a lower percentage of patients taking another anti-TNF drug – infliximab. In addition, a negative effect of taking vedolizumab and prednisone was found (Tables II–IV).
Table II
Comparison of selected variables between subjects with and without IL-2
[i] N and % were given for qualitative variables; median with quartile 1 and 3 were given for quantitative variables. Dependencies between qualitative variables were analysed with chi-square test or with Fisher’s exact test1. Differences in the level of quantitative variables were analysed with Mann-Whitney’s U test.
Table III
Univariate logistic regression models for IL-2
Table IV
Multivariate logistic regression models for IL-2
IL-6
In the group of patients with the presence of IL-6, a statistically significantly higher median CDAI index was found (158.5 vs. 108.6; p = 0.042). However, it should be emphasised that both these values are within the limits considered as clinical remission. No other relationships were found in this group.
IL-8
No significant dependency was detected between the presence of IL-8 and any of the selected variables, also in univariate logistic regression models.
IL-10
In the group of patients with the presence of IL-10, there were more taking steroids: budesonide (10.8% vs. 1.2%; p = 0.032), prednisone (multivariate logistic regression models, OR = 3.09; p = 0.041), and thiopurines (OR = 2.63; p = 0.036). No other relationships were found.
TNF-α
In the group with the presence of TNF-α there was a greater proportion of men (72.7% vs. 50.7%; p = 0.030), fewer patients were treated with infliximab (27.3% vs. 56.0%; p = 0.004), but not with adalimumab, and there were more patients treated with vedolizumab (43.2% vs. 21.3%; p = 0.021). These data were confirmed in univariate and multivariate logistic regression models (Tables V–VII).
Table V
Comparison of selected variables between subjects with and without TNF-α
[i] N and % were given for qualitative variables; medians with quartile 1 and 3 were given for quantitative variables. Dependencies between qualitative variables were analysed with chi-square test or with Fisher’s exact test1. Differences in the level of quantitative variables were analysed with Mann-Whitney’s U test.
Table VI
Univariate logistic regression models for TNF-α
Discussion
To the best of our knowledge, this is the first study that attempts to assess the immune system response, expressed by multiple cytokine concentrations, to asymptomatic SARS-CoV-2 infection in a group of patients with IBD, and possibly in any other group.
In our study, we assessed the concentration of 15 cytokines in a group of patients with IBD and asymptomatic SARS-CoV-2 infection defined by a positive serological test. None of the patients had symptoms of COVID-19 and most were in clinical remission from IBD. We found a higher concentration of IL-2 in the group of patients with a recent infection, which was expressed in the relationship between the concentration of this cytokine and the concentration of SARS-CoV-2 antibodies in the IgA + IgM class.
We found no relationship between other cytokines and asymptomatic SARS-CoV-2 infection.
In our study, there was no effect from the type and severity of IBD and the treatment applied on the course of the early phase of SARS-CoV-2 infection. However, a relationship was observed between the treatment and the concentration of some cytokines. Thus, in the case of IL-2, a negative association with infliximab, vedolizumab, and prednisone was found. Interestingly, the effect for adalimumab was the opposite. As for IL-10, a positive correlation was found with the intake of steroids and thiopurines. In the case of the presence of TNF-α, the percentage of patients treated with infliximab was significantly lower, and the percentage of patients treated with vedolizumab was higher.
In our study, we included a wide range of cytokines. These included those thought to be part of the innate immune system, but also the adaptive and chemokines, pro- and anti-inflammatory. It should be emphasised that a characteristic feature of cytokines is their pleiotropy, i.e. the ability of each of them to affect many different cells and show different actions. Therefore, qualifying a particular cytokine as appropriate for a given function is difficult and is based on the main direction of action.
Research to date has focused on understanding the role of the immune system in severe COVID-19. In many studies, a significant stimulation of the immune system was found, which is reflected, among others, in a significant increase in the concentration of pro-inflammatory cytokines. This phenomenon is called a cytokine storm.
A study by Qin et al. [12] found significantly elevated levels of IL-6, IL-8, IL-10, and TNF-α in patients with severe COVID-19 compared to patients with a milder course. The concentration of these cytokines, among many other factors, was associated with a higher risk of respiratory failure and death.
Similarly, Del Valle et al. [13] found elevated levels of IL-6, IL-8, and TNF-α in patients hospitalised for COVID-19. Moreover, high concentrations of these cytokines at admission was a prognostic factor.
In our study, we did not observe elevated levels of these cytokines. The asymptomatic course of COIVD-19 did not cause significant stimulation of the immune system or this stimulation was inhibited by anti-inflammatory mechanisms effectively and in the early stages. This may be shown by the elevated concentrations of IL-2 observed in our study. This cytokine is one factor responsible for regulating the activity of T lymphocytes, and it protects against autoimmunity; it is a strong growth factor of regulatory T lymphocytes. This is probably not the only mechanism inhibiting the development of excessive stimulation of the immune system leading to the symptomatic form of COVID-19, but it should be considered, because it is noticeable even in patients with asymptomatic forms of SARS-CoV-2 infection.
However, the role of IL-2 in COVID-19 infection is not clear. In the Fawzy et al. [14] study, high concentrations of this cytokine were also found in patients with severe COVID-19. Other authors (Kalfaoglu et al. [15], Silva et al. [16]) postulate dysfunction of IL-2 as a result of dysregulation of T lymphocytes in severe COVID-19 infection. As a result, the regulatory function of these cytokine decreases and the activation function of NK cells, T helper, and B lymphocytes increases.
Our work has some limitations. The diagnosis of SARS-Cov-2 infection is based on serological tests that were available in the initial phase of the pandemic. As we know, serological diagnostics is not the optimal tool for diagnosing a past SARS-CoV-2 infection, but due to its simplicity, it is most often used for this purpose. In our study, the time since infection could not be accurately determined because all patients were asymptomatic. The study group was relatively small, which could have resulted in too few patients with the presence of many cytokines, and thus the lack of reliable statistical analysis.
Our study is the first to assess the impact of asymptomatic SARS-CoV-2 infection on the immune system of IBD patients. We found a positive correlation between early infection markers (IgA + M antibodies) and the presence of IL-2. Immune disorders mediated by this cytokine have already been described in severe infections. Its impact on the course of COVID-19, and especially its role in the progression of the infection towards a severe form, requires further research.
