Randomized, placebo-controlled trial on clinical and immunologic effects of probiotic containing Lactobacillus rhamnosus R0011 and L. helveticus R0052 in infants with atopic dermatitis

Abstract

Background and aim: A beneficial role of probiotics, especially Lactobacillus rhamnosus, in children with atopic dermatitis (AD) has been reported. Their effects are primarily mediated through immune pathways, which need further investigation. This study aimed to investigate clinical and immunologic effects of probiotics in the treatment of children with AD. Methods: Thirty children with AD received probiotic Lacidofil and 28 other children with AD received placebo, one capsule per day. All children used emollient Trixera. SCORAD, specific IgE, specific IgG4 to cow's milk, CD45RA+, CD45RO+ and transitional CD45RA+RO+ CD4+ T cells were studied at day 1 and day 30. Results: Marked reduction of SCORAD in 63.3% of patients in the treatment group and in 32.1% in the placebo group was reported. The decline of SCORAD in patients who did not use topical steroids during the follow-up period was significant in the treatment group (p < 0.01) and not significant in the placebo group. After the treatment, levels of specific IgG4 to cow's milk increased (p < 0.001) and percentages of transitional recently activated CD45RA+RO+ T cells decreased (p < 0.05). Conclusion: The use of probiotic Lacidofil was associated with a higher number of patients who achieved marked clinical improvement and higher corticosteroid-sparing effect. Synergistic action of Lacidofil and emollient Trixera was also confirmed by significant decline of transitional recently activated CD45RA+RO+ CD4+ T cells. We found significant increase of the levels of specific IgG4 to cow's milk in the group of patients who received probiotic and emollient that was not reported in the treatment group and may be considered as a shift to tolerance.

Key words::

• Emollients
• T lymphocyte

Introduction

Atopic dermatitis (AD) (synonym atopic eczema) is a chronic inflammatory skin disease. Skin lesions result from aberrant immune response coupled with abnormal cutaneous reactivity. AD is a very common disease and represents a major public health problem worldwide. In the last decades the prevalence of AD has increased markedly. As many as 20% of preschool children in Western Europe have AD (1). Currently, there is no efficient treatment for AD. According to the International Consensus Conference on Atopic Dermatitis (ICCAD II), treatment of a patient with AD is based on adequate combination of auxiliary basic therapy and anti-inflammatory preparations, and also on discovering inducing factors and if possible their elimination (2). Beneficial effects of probiotics, especially Lactobacillus rhamnosus, in children with AD has been reported (3).

Probiotics are ‘helpful’ human bacteria that provide a variety of health benefits. Scientific literature on probiotics has increased remarkably in the last 10 years. Their mechanisms of action have been investigated experimentally. Studies indicate that probiotics can act by competing with pathogens, interacting and modulating local and systemic inflammatory and immune response, among others.

Meta-analysis of 10 randomized controlled trials describing the efficacy of probiotics in AD published in January 2008 showed an overall statistically significant difference favoring probiotics compared with placebo in reducing the SCORAD (4). Another review concluded that probiotics, especially L. rhamnosus GG, seem to be effective for the prevention of AD. Probiotics were also found to reduce the severity of AD in approximately half of the randomized placebo-controlled trials evaluated, although they were not found to change significantly most of the inflammatory markers measured in the majority of the evaluated trials (5). In contrast, other studies showed no therapeutic effect of probiotics against AD (6,7).

Understanding how lactobacilli act to prevent eczema requires further investigation (8). Prescott et al. noted that improvement in AD severity with probiotic treatment was associated with significant increases in the capacity for Th1 interferon (IFN)-gamma responses and altered responses to skin and enteric flora, and that effects of probiotics may be mediated through pathways that need to be explored further (9).

CD4+CD45RO+ cells in AD patients are present in skin lesions and supposed to play a crucial role in AD mechanisms (10–13). It was reported that the naïve (CD45RA+) to memory (CD45RO+) T-cell transition is characterized by a stepwise, unidirectional progression through CD45RA+RO+ transitional T cells (14). These cells can express cutaneous lymphocyte antigen (CLA) and showed E-selectin-binding ability, which are important for skin homing (15). Recently we demonstrated that significant decline of naïve-to-memory-transitional CD4+ cells is associated with marked improvement of AD in children (16).

We decided to study clinical and immunologic effects of probiotics in the treatment of children with AD. Initially, we asked the question regarding the choice of probiotic and inclusion criteria for patients. Despite the hypothesis that the exact probiotic strain used is less important than an adequate dose (17), it was reported that supplementation with L. rhamnosus, but not other probiotics such as Bifidobacteria animalis subsp. lactis, substantially reduced the cumulative prevalence of eczema (8). Moreover, it was shown that application of several probiotic microorganisms is not only inefficient but may lead to Th2 response, which supports allergy (18). Sistek et al. reported that combination of L. rhamnosus and B. animalis subsp. lactis improved AD only in food-sensitized children (19). A positive effect from probiotics treatment of AD was discovered in infants with cow's milk allergy (18). Meanwhile, it was also shown that children with cow's milk allergy can present sensitivity to probiotics. For that reason probiotic use in patients with cow's milk allergy has to be limited to products that do not contain milk (20). The majority of studies supporting a positive effect of probiotics in AD treatment were done on small children with AD (9,21–23). When we summarized all these facts we decided that positive clinical results from treatment of AD with probiotics could be achieved in small children with cow's milk allergy and probiotic should preferably contain a single strain of L. rhamnosus and should not contain milk. After studying the list of probiotic preparations registered in Ukraine it was determined that only Lacidofil (Institut Rosell Inc., Montreal, Quebec, Canada) contains 95% of L. rhamnosus. Moreover, Lacidofil is one of a few probiotic preparations without milk admixtures, because lactobacilli for this preparation are grown in milkless media. In our previous study we have shown significant improvement of clinical signs of AD children with cow's milk allergy after 1 month's use of Lacidofil, emollient cream Trixera, and emollient bath Trixera followed by significant increase of specific IgG4 to cow milk (24).

Material and methods

Subjects

We studied 58 children (35 males and 23 females) with AD up to the age of 4 (minimal age 2 months) with allergy to cow's milk confirmed by anamnestic data and specific IgE and/or specific IgG. The diagnosis of AD was made using Hanifin and Rajka criteria (25). Only patients who had no problems with their health other than AD were included in the study. All patients had the active phase of pathologic process. Disease severity was estimated by the same dermatologist using SCORAD before and after the treatment (26). Thirty children with AD received the probiotic preparation Lacidofil containing 95% L. rhamnosus R0011 and 5% L. helveticus R0052) one capsule per day (one capsule of preparation contains 2 billion viable lyophilized bacteria) for 30 days. The other 28 children with AD received placebo (malto-dextrin) one capsule per day. The children were randomized into two groups by their appearance at the clinic (the first patient to the treatment group, the second to the placebo group, etc.). During the follow-up period (30 days) all children used Trixera emollient cream and Trixera emollient preparation for bath (Laboratories Dermatologiques Avene, Paris, France) as moisturizers. Data from 13 non-atopic, healthy children of the same age who underwent blood tests before vaccination were used as controls. Data on the age and gender of patients and controls are presented in Table I.

Table I. Gender and ages of AD patients and controls.

Parameter	Treatment group (n = 30)	Placebo group (n = 28)	Normal controls (n = 13)
Males	18	17	8
Females	12	11	5
Median age (range) in months	18.43±11.94 (2–46)	18.21±10.51 (2–44)	18.23±11.42 (2–43)

Ethical permission for the study was granted by the local ethic research committee. Informed consent forms were received from the parents.

Blood collection procedure

Blood collection was done using aspiration by monovettes (Sarstedt, Germany). The first was with Li-Heparin for lymphocyte subset analysis by flow cytometry and the second was without reagents to prepare serum samples.

Flow cytometry of lymphocyte subsets

Lymphocyte subsets were identified by three-color flow cytometry using an erythrocyte-lysing whole blood method of lymphocyte staining by FITC-, PE- or PE-Cy5-conjugated monoclonal antibodies to the following human lymphocyte subsets: CD3 + CD4+ CD45RA+, CD3 + CD4+CD45RO+, and CD4+ CD45 RA+RO+ (Becton Dickinson, San Jose, CA, USA). Stained samples were analyzed by FACScan flow cytometer using FACScan Research & Lysis software (Becton Dickinson). For a defining of the lymphocyte gate, whole blood samples were incubated with LeucoGATE (anti-CD45-FITC and anti-CD14-PE monoclonal antibodies, Becton Dickinson) to permit discrimination of lymphocytes from granulocytes, monocytes, debris, and unlysed or nucleated red blood cells. As background control, whole blood samples were incubated with SimulTEST control (IgG1-FITC + IgG2-PE, Becton Dickinson). Analyses were initiated within 2 h after the collection of venous blood.

Specific IgE and specific IgG4 to cow's milk in the sera of AD patients were studied by ELISA using a commercial kit produced by Dr Fooke Laboratories GmbH, Germany.

All studies were carried out on day 1 and day 30.

Statistical methods

Data were presented as mean ± standard deviation of the mean. Wilcoxon matched pairs test, Fisher's exact test, and unpaired t test with Welch correction were used. The results were considered significant if p < 0.05.

Results

After the treatment SCORAD decreased significantly from 34.2±3.8 to 19.8±2.2 (p < 0.01) in the treatment group and from 36.9±4.0 to18.8±3.3 (p < 0.01) in the placebo group. Marked reduction of SCORAD in 19 patients from the treatment group and in 9 patients from the placebo group was reported. Thus, the number of patients with a marked decline of clinical signs was higher in the treatment group (63.3%) than in the placebo group (32.1%) (p = 0.02). During the follow-up period three (10%) children from the treatment group and nine (32.1%) children from the placebo group had to use topical steroids at least once. The decline of SCORAD in patients who did not use topical steroids during the follow-up period was significant in the treatment group (decreased from 30.6±3.7 to 20.9±2.3, p < 0.01) and was not significant in the placebo group (decreased from 27.2±4.1 to 23.2±4.3).

Levels of specific IgE to cow's milk in serum of children with AD did not differ significantly before and after the treatment in both groups. Levels of specific IgG4 to cow's milk after the treatment increased significantly in the treatment group in comparison with day 1 (Table II).

Table II. Levels of specific IgE and specific IgG4 to cow's milk in serum of patients from study and control groups at day 1 and day 30.

	Treatment group (n = 30)		Placebo group (n = 28)
Parameter	Day 1	Day 30	Day 1	Day 30	Non-atopic controls (n = 13)
sIgE to cow's milk (IU/ml)	8.4±49.55	6.39±3.38	9.04±4.08	15.30±7.05	< 0.35
sIgG4 to cow's milk (μg/ml)	128.35±21.56	141.57±23.09*	195.44±24.10	209.90±20.71	< 10

*p < 0.001 (Wilcoxon matched pairs test).

Percentages and absolute counts of CD45RO + memory-effector T cells were increased before and after the treatment in both treatment and placebo groups compared with the control group. After the treatment, percentages of transitional recently activated CD45RA+RO+ T cells in the treatment group decreased significantly in comparison with day 1 and with normal controls (Table III).

Table III. Percentages of lymphocyte subsets in peripheral blood of patients from study and control groups and non-atopic controls at day 1 and day 30.

	Treatment group (n = 30) (%)		Placebo group (n = 28) (%)
Lymphocyte subset	Day 1	Day 30	Day 1	Day 30	Non-atopic controls (n = 13) (%)
CD4CD45RA+	59.88±6.98	54.69±7.41	41.77±12.10	36.67±10.46†	64.14±4.38
CD4CD45RA+RO+	19.37±1.66	13.39±1.34*†	19.74±3.46	14.01±2.13	21.22±3.32
CD4CD45RO+	42.77±4.42†	45.83±4.72†	51.86±8.03†	55.32±7.18†	23.26±2.72

*p < 0.05 in comparison with levels at day 1 (Wilcoxon matched pairs test).

†p < 0.05 in comparison with non-atopic controls (unpaired t test with Welch correction).

Discussion

In our study more patients treated with Lacidofil achieved satisfactory clinical results with less episodes of the use of topical corticosteroids than those who received placebo. The emollient treatment can reduce topical corticosteroid consumption in infants with AD (27,28). In our study children with AD who were treated with emollient and Lacidofil had higher corticosteroid-sparing effect than those treated with emollient and placebo. Thus Lacidofil could reinforce the corticosteroid-sparing effect of emollients.

The role of IgG4 in food allergy is controversial (29). According to the European Academy of Allergology and Clinical Immunology Task Force Report, IgG4 should not be considered as a factor that induces hypersensitivity, but rather as an indicator for immunological tolerance linked to the activity of regulatory T cells (30). Food-sensitized infants with high levels of IgG4 to food allergens are more likely to consume these foods at the age of 4 years than infants with low IgG4 levels (31). It was also shown that maintenance of tolerance to cow's milk in atopic children and adults without cow's milk allergy is associated with elevated levels of specific IgG4, in combination with low specific IgE (32). In this context we speculate that the significant increase of specific IgG4 levels in the study group that was not reported in the control group could be considered as a shift to tolerance.

In addition, there is a lack of information about recently activated CD45RA+RO+ T cells. Its transitional status between naïve and memory-effector T cells, expression of skin homing markers, and correlation with AD severity confirmed its involvement in the pathologic process of AD. Moreover, significant decline of naïve-to-memory-transitional CD4 + cells is associated with marked improvement of AD in children (16). We speculate that transitional recently activated CD45RA+RO+ cells in children with AD should be noticeably lower than those of healthy children during AD remission, with subsequent elevation of its rate during relapses. In the present study the levels of naïve-to-memory-transitional CD4+ cells had the tendency to decrease after the treatment in both groups but significant decline of recently activated CD45RA+RO+ T cells was found only in the treatment group that could be explained by combined effect of Lacidofil and emollient treatment in comparison with the effect of emollient alone in children with AD. It is also supported by the higher number of patients with marked decline of SCORAD in the treatment group reported after the treatment.

Probiotics and emollients have different modes of action (33) and could be easily combined with each other and with topical steroids. In our study we have shown that probiotic Lacidofil and emollient Trixera have a synergistic action in children with AD. More patients obtained better response and less topical corticosteroids were required to achieve satisfactory clinical response in the group with combined treatment. Such a treatment strategy is beneficial because it may decrease the side effects of topical steroids. It is especially important for AD as for chronic skin disease that both preparations could be used for long periods of time without side effects (33,34). It is also possible to use probiotics like emollients even in the remission phase of AD. Further studies are important to investigate dose-dependent effects of probiotic Lacidofil and its influence on the frequency of AD flares in long-term perspective.

This study has shown that the use of the probiotic preparation Lacidofil was associated with a higher number of patients who achieved marked clinical improvement and higher corticosteroid-sparing effect. Synergistic action of Lacidofil and emollient Trixera was also confirmed by the dynamics of transitional recently activated CD45RA+RO+ CD4+ T cells. We found a significant increase in the levels of specific IgG4 to cow's milk in the group of patients who received probiotic and emollient that was not reported in the control group and may be considered as a shift to tolerance.

Acknowledgements

The authors report no conflicts of interest.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.