TLR3 and TLR7 prognostic role in early pediatric rotavirus infection

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Introduction

Rotavirus infection retains a leading position among acute intestinal infections (AKI) in children under the age of 5 years, not only in Russia, but also worldwide, despite the introduction of vaccination against this disease in a number of countries [1, 2]. According to Rospotrebnadzor, the incidence rate of rotavirus infection in our country in 2021 was 49.77 per 100 thousand population, which is 46.6% more compared to the previous year. The incidence is especially high among children under the age of 1 (593.48 per 100 thousand) and 1-2 years (890.49 per 100 thousand), while the vaccination coverage of the target cohort was only 6.23% [3]. The statistical data provided indicate the high medical and social significance of rotavirus infection for the healthcare of many countries, including the Russian Federation.
In the pathogenesis of rotavirus infection, the leading role is played by the direct cytopathic effect of the pathogen on mature enterocytes with the development of atrophy of their villi and the death of cells of the mucous membrane of the small intestine. The process of introduction of rotavirus begins with overcoming the protective layer of mucus, interaction with the molecules of the cell membrane of enterocytes (sialic acid, integrins, etc.) and intestinal microbiota, which ultimately leads to damage to the mucous membrane. At the same time, enterocytes, resisting infection, use a number of factors of innate immune protection to prevent rotavirus invasion, one of which is the transmission of a toll-like receptor (TLR) signal [1].
TLRs are among the leading structures of innate cellular immunity that recognize various pathogens, including rotaviruses. TLR-3 and TLR-7 play the greatest role in determining rotavirus RNA. Their activation coordinates a long-term adaptive immune response to an infectious agent. However, activation of pro-inflammatory cytokines is possible, which ultimately leads to damage to enterocytes. Activation of TLR-3 also induces chemokines and type I interferon. In an experiment on mice that lacked TLR-3, a decrease in the production of type I interferon and a higher viral load during rotavirus infection were found compared with wild mice [4]. The TLR-3 inhibitor norcurarinol (lavandulylated flavone isolated from the roots of the Sophora flavescens plant) is capable of suppressing cytopathic effects caused by rotavirus and affecting its replication, which opens up new prospects in the search for effective etiotropic drugs for the treatment of rotavirus infection [5].
Studies conducted to date have shown that TLR-dependent signaling in dendritic cells is involved in the secretion of type I interferon in rotavirus infection. Infection of plasmocytoid dendritic cells with rotavirus leads to endosome-dependent (and possibly TLR-7-mediated) type I interferon secretion, which is triggered by viral double-stranded RNA. The central role of TLR-dependent protection against rotavirus is also indicated by the fact that the absence of MyD88 (an important adapter protein involved in the transmission of signals from various TLRs) leads to an increase in the virulence of this pathogen, the severity of diarrhea and a violation of humoral immunity [2].
The ability of TLR-3 to recognize rotavirus and, thus, to form an immune response is associated with an increase in the expression of TLR-3 in the intestine. Since rotavirus infection is usually more severe in early childhood, which coincides with a lower level of TLR-3 expression in this group of patients compared to adults, one of the possible reasons for this is the increased transmission of TLR-3 signals with age in enterocytes [6]. It was found that low expression of TLR-3 contributes to susceptibility to rotavirus infection [7]. TLR-3 and TLR-7 affect the production of IgA class antibodies and seroconversion to the Rotarix vaccine [8]. Consequently, these types of receptors can play an important role not only in the course of rotavirus infection, but also to a certain extent determine the outcomes of this disease in children.
The aim of the study was to determine the value of TLR-3 and TLR-7 in young children with rotavirus infection as predictors of the unfavorable course and outcomes of the disease.

Materials and methods

Under our supervision, 20 children from 1 month to 3 years with rotavirus infection of moderate severity, who were divided into several groups depending on the initial number of immunocompetent ones, were under our supervision in the GBUZ "DGKB No. 9 named after G.N. Speransky" DZM and GBUZ "DGKB named after Z.A. Bashlyaeva" DZM in 2020-2021. cells expressing TLR-3 and TLR-7. These cells were determined on the 1st day of hospitalization by the method of precision cytometry on the NaviosTM BC device using Beckman Coulter reagents by employees of the Department of Clinical Laboratory Diagnostics of the Academic Educational Center of the Russian Ministry of Health (Prof. S.A. Lugovskaya, Associate Professor E.V. Naukova, Associate Professor M.E. The Postman). The average levels of the number of immunocompetent cells expressing TLR-3 and TLR-7 in practically healthy children aged 1 month to 3 years were determined by them earlier (0.6 (0.5-0.7) and 0.7 (0.6-0.8), respectively, in absolute terms).
The diagnosis of rotavirus infection was established by detecting rotavirus RNA in the feces by PCR and the antigen of this microorgasm by immunochromatography in the laboratories of these stations. In addition, all patients with rotavirus infection underwent a general clinical dynamic examination, clinical blood and urine tests, biochemical blood analysis, ionogram, determination of the acid-base state (CSF), bacteriological stool culture, a co-program, ultrasound of the abdominal cavity, according to the indications, patients were consulted by an allergist, an otorhinolaryngologist, pulmonologist. After discharge from the hospital, the children were followed by catamnestic observation for 6-12 months, during which the nature of the stool, the presence of pathological impurities in it, the number of episodes of acute respiratory infections (ARI), allergic diseases and pathology from the gastrointestinal tract (gastrointestinal tract) were recorded.
Concomitant severe somatic diseases at the stage of decompensation, hereditary genetic diseases, immunodeficiency conditions, as well as taking anti-bacterial and/or immunomodulatory drugs, probiotics 1 month before the present study were excluded in all the examined children. Patients with rotavirus infection received complex treatment according to officially approved standards and clinical recommendations, as well as an additional preparation of human recombinant 2b-interferon (VIFERON ® , Pheron LLC, Russia) for 150 thousand IU 2 times/day with a course of 5 days rectally. The research protocol is approved by the local Ethics Committee.
Quantitative features were expressed in the form of the average value of the indicators (M) and the standard error (m). Statistical processing of the obtained results was carried out using the STATISTICA 8.0 program for independent samples using the nonparametric Mann-Whitney criterion and the Pearson criterion χ² .

Results

Based on the number of immunocompetent cells expressing TLR-3 (CD3+/TLR3+), the patients were divided into 2 groups - with low (17 people) and high (3 people) levels. The duration of the main clinical symptoms of the disease in them is shown in Figure 1.

Figure 1. Duration of clinical symptoms of rotavirus infection in children with different levels of TLR3-expressing immunocompetent cells

It turned out that the duration of fever, intoxication, reduction of soft tissue turgor, thirst, flatulence, diarrhea and oliguria in both groups did not differ significantly. Vomiting was observed 2 times longer in patients with low CD3+/TLR3+ (1.6 (1.4-1.8) vs. 0.7 (0.0-1.4) days), but this difference was not significant. Symptoms such as dry lips, tongue and skin persisted significantly longer in the same group (respectively, 2.3 (2.2-2.4) vs. 1.3 (1.0-1.6) and 2.0 (1.9-2.1) vs. 1.3 (1.0-1.6) days at p<0.05 according to the Pearson criterion and the criterion Manna-Whitney). At the same time, abdominal pain was characteristic only for children with a high content of TLR-3 expressing cells during 3.0 (0.0-6.0) days. Attention was drawn to the fact that in the group of patients with a low CD3+/TLR3+ content, the maximum frequency of stools was slightly less (5.8 (4.7-6.9) versus 7.7 (5.5-9.9) times/day), and the maximum frequency of vomiting and the severity of fever was greater (respectively, 6.3 (5.3-7.3) versus 1.7 (0.0-3.4) times/day and 38.3 (38.1-38.5) versus 37.1 (36.9-37.3)oS) compared with the group of children in whom the number of immunocompetent cells expressing TLR-3 was high, although these differences were not significant. There were no significant differences between the groups in the hemogram, although there was a slight predominance of the total number of leukocytes and neutrophils with a slightly smaller number of lymphocytes in patients with a low CD3+/TLR3+ content relative to patients with a high content of cells expressing TLR-3 (Table 1). The number of monocytes in both groups was almost the same.

 

Given the longer persistence of signs of exicosis in the group of children with a low CD3+/TLR3+ content, it is quite logical that the duration of infusion therapy in them, conducted for the purpose of rehydration and detoxification, was significantly higher compared to children in whom the number of immunocompetent cells expressing TLR-3 was high – 3.0 (2.7-3.3) versus 0.7 (0.0-1.4) days (p0.05 according to the Pearson criterion and the Mann-Whitney criterion). Also, only in this group, antibacterial therapy was added to the treatment for 2.1 (1.4-2.8) days due to the addition of bacterial microorganisms to the rotavirus, which was expressed in the appearance of pathological admixture of greenery and mucus in the feces on 2-3 days from the moment of hospitalization in patients, the preservation of fever.
Estimating the number of CD3+/TLR7+ in the same 20 children we observed with rotavirus infection, they were also divided into 2 groups depending on the level of TLR-7 expression - low (15 people) and high (5 people). The duration of the main clinical symptoms of the disease in them is shown in Figure 2.

Figure 2. Duration of clinical symptoms of rotavirus infection in children with different levels of TLR7-expressing immunocompetent cells

As can be seen, the duration of fever, intoxication, signs of exicosis and symptoms of gastrointestinal tract damage in both groups turned out to be practically identical. Only abdominal pain was observed only in patients with a high content of immunocompetent cells expressing TLR-7 – 3.0 (0.0-6.0) days (p0.05 according to 2 Pearson criterion and Mann-Whitney criterion). The maximum frequency of stool (6.0 (4.8-7.2) vs. 6.4 (4.4-8.4) times /day) and vomiting (5.9 (4.7-7.1) vs. 4.6 (3.0-6.2) times/day), as well as the severity of fever (38.0 (37.8-38.2) vs. 38.4 (37.8-39.0)oS) did not differ significantly in both groups. In the hemogram of children of this group, the number of monocytes was slightly lower than normal and significantly lower compared to patients whose CD3+/TLR7+ content was low (Table 2).

 

The duration of infusion therapy was found to be almost a day longer in children with a low content of immunocompetent cells expressing TLR-7 compared to patients with a high content - 2.9 (2.5-3.3) versus 2.0 (1.1–2.9) days (the differences are unreliable). At the same time, antibacterial agents were added for 2.5 (1.8-3.2) days to complex treatment, including interferon therapy, only for children with a low CD3+/TLR7+ content due to the development of mixed infection due to the addition of bacterial pathogens, as evidenced by the appearance of green and mucus impurities in the stool (p<0.05 according to b2 Pearson criterion and Mann-Whitney criterion).
After the acute phase of the disease was stopped, patients were discharged from the hospital, and catamnestic observation was established for them for 6-12 months, the results of which are presented in Table 3.

 

It was found that only in children with a low content of immunocomponent cells expressing both TLR-3 and TLR-7, repeated episodes of acute respiratory infections and cases of allergic diseases (atopic dermatitis, food allergy) were recorded during the period of catamnestic observation. Gastrointestinal pathology in the form of reactive pancreatitis was significantly more often formed in children with a low CD3+/TLR3+ content.

Discussion

The results obtained in this study indicate the important role of TLR-3 and TLR-7 in the course and outcomes of rotavirus infection in young children. In particular, it has been shown that the disease proceeds with more pronounced symptoms of exicosis in patients with a low content of immunocompetent cells expressing TLR-3. The fact that abdominal pain was observed only in children with a high content of CD3+/TLR3+ and CD3+/TLR7+, in our opinion, is not a characteristic feature, but is most likely due to the small number of patients in these groups. The same circumstance can probably explain the lower number of monocytes in the hemogram in children with a high content of cells expressing TLR-7, compared with patients who had a high number.
It is also important that the duration of infusion therapy was significantly longer in children with a low CD3+/TLR3+ content, consistent with the severity of their exicosis, as mentioned above. Another feature of the treatment was the need to add antibacterial agents to the complex of therapeutic measures for patients with a low content of immunocompetent cells expressing TLR-3 and TLR-7, due to the addition of bacterial flora to rotavirus on 2-3 days from the moment of hospitalization, which was expressed in the appearance of greenery and mucus in the feces, the preservation of fever.
Catamnestic observation for 6-12 months of children who had rotavirus infection demonstrated that an unfavorable course of this period occurred in groups that had low CD3+/TLR3+ and CD3+/TLR7+. It can be assumed that both the more severe course of the disease in the acute period and the non-smooth course in the catamnesis with the formation of unfavorable outcomes from the gastrointestinal tract in children with in these groups is due to a significant viral load relative to patients with a high content of immunocompetent cells expressing both types of TLRs studied [4].
There is no doubt that the age factor plays an important role in the course and outcomes of rotavirus infection, as it was noted for young children by J. Pott and co-authors, who emphasized a lower level of TLR-3 expression in them compared to adults [6]. The "vicious circle" closes as a result of the fact that low expression of TLR-3 contributes to susceptibility to rotavirus infection [7].

Conclusion

Based on the data obtained in the study, an initially low number of immunocompetent cells expressing TLR-3 and TLR-7 can be considered predictors of the unfavorable course and outcomes of rotavirus infection in young children, which must be taken into account when determining the tactics of treatment of this disease in this group of patients, including, if indicated, not only interferon therapy, but also and antibacterial agents.
Conflict of interest
The research was carried out with the financial support of Feron LLC, which was given the opportunity to review the preliminary version of this publication for factual accuracy, but the authors are fully responsible for the final content and interpretation.

About the authors

Sergey G. Gorbunov

Russian Medical Academy of Continuig Professional Education of the Ministry of Healthcare of the Russian Federation

Email: gsgsg70@mail.ru
ORCID iD: 0000-0001-6335-0487

DSc (Medicine) Head of the Department of Children's Infectious Diseases, Professor of the Department of Children's Infectious Diseases

Russian Federation, 123995, Moscow, Barrikadnaya str.,2/1

Ludmila N. Mazankova

Russian Medical Academy of Continuig Professional Education of the Ministry of Healthcare of the Russian Federation

Email: mazankova@list.ru

DSc (Medicine), Professor, Head of the Department of Children’s Infectious Diseases

Russian Federation, 123995, Moscow, Barrikadnaya str.,2/1

Alexandr N. Os'kin

Russian Medical Academy of Continuig Professional Education of the Ministry of Healthcare of the Russian Federation

Author for correspondence.
Email: o_alksandr@mail.ru

PhD Student, Department of Children’s Infectious Diseases

Russian Federation, 123995, Moscow, Barrikadnaya str.,2/1

References

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