# Abstract

 

## Background Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in infancy with a complex pathology. In adults, the clinical severity of AD has been associated with increases in T helper cell type (Th) 2, Th22, and Th17 serum markers, including high levels of CC chemokine ligand (CCL) 17 and CCL22 chemokines. 

## Objective To explore the possible association between serum chemokine levels and AD severity in infants with moderate-to-severe AD and elevated immunoglobulin E (IgE). 

## Subjects and methods Serum samples ( *n* = 41) obtained from a randomized, double-blind, and clinical dietary intervention study were used to study biomarkers in infants with AD. Baseline- and post-intervention samples (4 months) were used, six chemokines and nine ratios thereof were analyzed using Luminex and correlated to AD severity. In the initial study, the infants were randomized to receive extensively hydrolyzed whey-based formula without (control) or with short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (9:1) and *Bifidobacterium breve* M-16V (active). 

## Results 31 Infants up to 11 months of age, with an objective-SCORAD score (oSCORAD) ≥ 20 and elevated total-IgE and/or specific-IgE levels were included. In time, the median oSCORAD decreased in both groups by −8 (control, *p*  \< 0.05; active, *p*  \< 0.01). Irrespective of dietary intervention, several changes in Th2 chemokines (CCL17 and CCL22), inflammatory chemokine (CCL20), and the Th1 chemokine, CXC chemokine ligand (CXCL) 9, were detected over time. Overall CCL17 correlated to oSCORAD ( *r* = 0.446, *p*  \< 0.01). After 4 months of dietary intervention, CXCL9 was higher ( *p*  \< 0.01) in the active group compared with control [active, 2.33 (1.99--2.89); controls, 1.95 (1.77--2.43) log 10 median (range)]. In addition, a reduction in Th2/Th1 chemokine ratios for CCL17/CXCL9, CCL22/CXCL9, CCL20/CXCL10, and CCL20/CXCL11 was detected associated with the active intervention. 

## Conclusion While this study is small and exploratory in nature, these data contribute to immune biomarker profiling and understanding of AD in infants. # Results 

## Infant Characteristics Overall, 53 infants with moderate-to-severe AD were screened. Of these, 31 infants had elevated IgE levels and were therefore considered eligible for randomization. In total, 26 infants completed the study (Figure [1](#) ). Baseline characteristics ( *n* = 31) are shown in Table [1](#) . <figure> <p><img src="" /></p> <figcaption>Flowchart of study inclusion and exclusion.</figcaption> </figure> ::::table-wrap

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Baseline characteristics of the clinical study ( *n* = 31).
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Characteristic Control group ( *n* = 15) Active group ( *n* = 16) *p*Values

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Male, *n* (%) 9 (60.0) 11 (68.8) 0.72
Age (months)\
Mean (±SD) 6.5 (1.8) 7.4 (2.4)\
Median (Q1--Q3) 6 (6--7) 8 (6--10) 0.15
Range 4--10 2--11\
Ethnicity, *n* (%) 0.82
Caucasian/White 11 (73.3) 13 (81.25)\
African/Black 1 (6.7) 0\
Asian 1 (6.7) 0\
Combination of above or other 2 (13.3) 3 (13.3)\
Gestational age (mean ± SD, weeks) 39.8 (1.2) 39.4 (1.8) 0.71
Birth weight (mean ± SD, g) 3,538.0 (428.6) 3,820.3 (718.2) 0.14
Delivery by cesarean section, *n* (%) 1 (6.7) 3 (18.8) 0.60
Length-for-age (mean ± SD, *z* -scores) −0.4667 (0.9155) 0.3125 (0.9465) **0.02**
Weight-for-age (mean ± SD, *z* -scores) −0.5333 (0.7432) 0.125 (0.8062) **0.03**
Head circumference-for-age (mean ± SD, *z* -scores) −0.2143 (1.369) 0.0625 (0.7719) 0.32
Started with breastfeeding, *n* 13 14\
Duration of exclusive breast feeding (mean ± SD, months) 1.5 (2.1) 3.0 (3.1) 0.14
Time of first introduction cow's milk protein (mean ± SD, months) 1.5 (2.1) 3.9 (4.3) 0.10
Time of first solid food introduction (mean ± SD, months) 5.3 (1.1) 5.2 (1.2) 0.86
oSCORAD score (mean ± SD) 26.53 (4.307) 24.94 (5.221) 0.22
Any topical corticosteroid used\
Yes, *n* (%) 14 (93.3) 15 (93.8) 1.00
Hydrocortisone acetate 1%, *n* 12 14 0.65
Triamcinolone acetonide 0.1%, *n* 5 4 0.70
Mometasone furoate 0.1%, *n* 1 2 1.00
Total immunoglobulin E (IgE)\
log 10 Mean (SD) 1.761 (0.4890) 1.562 (0.3508) 0.11
Total IgE log 10 min--max 1.000--2.963 0.8451--2.332\
Sensitization to aero-allergens 0.24\
Negative, *n* (%) 7 (46.7) 11 (68.8)\
Positive, *n* (%) 6 (40.0) 3 (18.8)\
Missing, *n* (%) 2 (13.3) 2 (12.5)\
Sensitization to food allergens 0.65\
Negative, *n* (%) 3 (20.0) 2 (12.5)\
Positive, *n* (%) 12 (80.0) 14 (87.5)\
Parental allergies: at least 1 positive allergic manifestations\
Mother (total 31), *n* (%) 6 (40) 6 (37.5) 1.00
Father (total 26), *n* (%) 7 (58.3) 7 (50) 0.71
Siblings in household (blood related)\
None 10 (66.7) 9 (56.3) 0.72
1 2 (13.3) 5 (31.3) 0.39
2 or more 3 (20) 2 (12.6) 0.65
Day care attendance \[yes, *n* (%)\] 11 (73.3) 12 (75.0) 1.00
Exposure to smoking in household \[yes, *n* (%)\] 5 (33.3) 4 (25.0) 0.70:::: The infants were well balanced over the study groups with respect to the baseline characteristics; only significant differences for length and weight were observed; infants in the control group were smaller in respect to length, weight. At baseline, the length-for-age *z* -scores for infants in the control group were significantly lower ( *p*  \< 0.05) than the active group [mean (SD) −0.4667 (0.9155) versus 0.3125 (0.9465), respectively]. The weight-for-age *z* -scores were also significantly lower ( *p*  \< 0.05) in the control group than the active group with a mean (SD) of −0.5333 (0.7432) and 0.125 (0.8062), respectively. Atopic dermatitis severity was classified as moderate-to-severe with a median oSCORAD of 23 (Q1--Q3 = 21--27). All infants were born at term with a median gestational age of 39 weeks, and only 12.9% of the infants were born *via* cesarean section. The mean age of infants at the time of inclusion was 7 months (median 6, range 2--11 months). The male/female ratio was approximately 2:1 in both study groups. The majority (80%) of infants were of Caucasian descent. At baseline, total IgE was comparable in both groups. In addition, 83.9% of the infants were sensitized to at least one food allergen; 29% to at least one aero-allergen, which was comparable in both groups. For all infants, topical corticosteroids were prescribed before screening for this study. The topical corticosteroids were used in 93.5% of the infants for a mean (SD) of 124.6 (73.1) days before start of dietary intervention. Topical corticosteroid use was evenly distributed between the study groups, with a median (Q1--Q3) of 135 (67--150) days of topical steroid use in the control group and 113 (65--182) days in the active group, with no significant differences observed. Low potency topical corticosteroids (hydrocortisone acetate 1%) were most commonly used (83.9%) compared with moderate potency triamcinolone acetonide 0.1% (29%) and high potency mometasone furoate 0.1% (9.7%). The median [interquartile range (Q1--Q3)] for exclusive breastfeeding before study enrollment was 0 (0--3) months in the control group and 3 (0--6) months in the active group, but this was not significantly different. Cows' milk protein was introduced at a median (Q1--Q3) of 0 (0--3) months and 3 (0--6) months in the control and active groups, respectively, ( *p*  > 0.05). Complementary feeding was first introduced at a median (Q1--Q3) of 5 (4--6) months among all infants. 

## Effect on Severity of AD At baseline, oSCORAD between the study groups was comparable, with a median (Q1--Q3) oSCORAD in the control group of 24 (21--27) and 21 (20--26) in the active group. After 4 months of dietary intervention, the average oSCORAD decreased in those infants ( *n* = 26) who completed the study. The median change in both groups of −8, resulted in a median (Q1--Q3) post-intervention oSCORAD of 17 (12--21) in the control group and 13 (12--17) in the active group. Interestingly, only four out of thirteen (31%) infants in the active group retained an oSCORAD above 15 (moderate AD) compared with eight out of thirteen (62%) infants in the control group. AD exacerbations occurred in 8 out of 26 infants (4 in both groups 30.8%) during dietary intervention period. The use of topical steroids during the dietary intervention period appears similar in both groups ( *p*  > 0.05) with a median (Q1--Q3) topical steroid use of 118.5 (113.5--120.8) days in the control group and 117.0 (113.0--121.5) days in the active group. 

## Exploratory Immune Profiles in AD In total, 41 serum samples obtained from the clinical study were used for exploring the biomarker profiles of infants with moderate to severe AD and elevated IgE levels. For these infants from which serum samples were obtained, reduction of oSCORAD at baseline and after 4 months of dietary intervention is shown in (Figure [2](#) ). Non-parametric analyses were performed and indicated within the figures. Small sample sizes are in general a good argument to use non-parametric statistics, as possible skewness in the data may not be revealed. Additional parametric analysis using ordinary one-way ANOVA was performed, to show robustness of the data. The analysis indicated no differences between the groups at baseline and confirmed the significant decline within control ( *p*  \< 0.001) as well as within active ( *p*  \< 0.0001) of oSCORAD, which support the non-parametric analysis. <figure> <p><img src="" /></p> <figcaption>Reduction of atopic dermatitis severity over time. Individual oSCORAD of the control (open dots) and active (closed triangles) groups at baseline and after 4 months of dietary intervention, for infants from which serum samples were available ( <em>n</em> = 41). Line represents median ± interquartile range. Significant differences within the groups are depicted with asterisks (non-paired W-test, * <em>p</em>  \< 0.05, \*\* <em>p</em>  \< 0.01).</figcaption> </figure> Several chemokines were measured in serum at baseline and at the end of intervention and correlated to AD severity. We detected an overall significant correlation between oSCORAD and CCL17 levels (Spearman *r* = 0.446, *p*  \< 0.01) and CCL22 (Spearman *r* = 0.288, *p*  \< 0.05) (Table [2](#) ). In addition, at baseline ( *n* = 18), a significant correlation was detected between levels of IgE and chemokines CCL17 (Spearman *r* = 0.721, *p*  \< 0.01) and CCL22 (Spearman *r* = 0.549, *p*  \< 0.05). Moreover, a significant correlation between galectin-9 and chemokines CCL20 (Spearman *r* = 0.585, *p*  \< 0.05) and CCL22 (Spearman *r* = 0.492, *p*  \< 0.05) was observed. ::::table-wrap
::: caption
Two-tailed Spearman correlation of chemokines and T helper cell type
(Th) 2/Th1 ratio to atopic dermatitis severity (oSCORAD) ( *n* = 41).
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Chemokine Spearman *r* *p*Value

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CC chemokine ligand (CCL) 17 0.446 **0.002**
CCL20 0.163 0.273
CCL22 0.288 **0.049**
CXC chemokine ligand (CXCL) 9 −0.177 0.234
CXCL10 0.034 0.824
CXCL11 0.042 0.780
CCL17/CXCL9 0.375 **0.009**
CCL17/CXCL10 0.318 **0.029**
CCL17/CXCL11 0.364 **0.012**
CCL20/CXCL9 0.239 0.105
CCL20/CXCL10 0.032 0.832
CCL20/CXCL11 0.032 0.832
CCL22/CXCL9 0.259 **0.079**
CCL22/CXCL10 0.187 0.207
CCL22/CXCL11 0.187 0.208:::: Total IgE and galectin-9 were measured in serum at baseline and at the end of intervention. Both levels, however, did not significantly change in either group (Figures [3](#) A,B). The levels of Th2 driving chemokines CCL17 and CCL22 significantly changed over time (Figures [3](#) C,D). In the control group, a significant reduction of CCL22, but not CCL17, was detected (W-test, *p*  \< 0.05), whereas in the active group, both CCL17 and CCL22 were significantly reduced (W-test, *p*  \< 0.05, for both). CCL20, a strong chemokine for DCs, remained unchanged in the control group, yet was significantly reduced in the active group after 4 months of dietary intervention (W-test, *p*  \< 0.05) (Figure [3](#) E). While no differences were observed in the release of Th1-driving chemokines CXCL9, CXCL10, and CXCL11 in the control group, after 4 months of dietary intervention, CXCL9 levels were significantly higher in the active group as compared with the control group (MWU, *p*  \< 0.01). The dietary intervention resulted in a higher CXCL9 level (log 10 median, 2.33; range 1.99--2.89) in the active group as compared with the control group (1.95; 1.77--2.43) (Figures [3](#) F--H). Moreover, additional parametric analysis using ordinary one-way ANOVA confirm our statistical findings within the serum biomarker analysis, although small sample size may not reveal possible skewness of the data and therefore the parametric analysis are not shown. <figure> <p><img src="" /></p> <figcaption>Allergy markers measured in serum of the control (open dots, <em>n</em> = 20) and active (closed triangles, <em>n</em> = 21) groups before and after dietary intervention. Immunoglobulin E (IgE) <strong>(A)</strong> , galectin-9 <strong>(B)</strong> , CC chemokine ligand (CCL) 17 <strong>(C)</strong> , CCL22 <strong>(D)</strong> , CCL20 <strong>(E)</strong> , CXC chemokine ligand (CXCL) 9 <strong>(F)</strong> , CXCL10 <strong>(G)</strong> , and CXCL11 <strong>(H)</strong> . Line represents median ± interquartile range. Significant differences within the groups are depicted with asterisks (non-paired W-test, * <em>p</em>  \< 0.05) and between groups (using Mann–Whitney <em>U</em> -test, \*\* <em>p</em>  \< 0.01).</figcaption> </figure> 

## Chemokine Ratios Change Over Time and due to Dietary Intervention Knowing that the immune balance between chemokines determines the type of cells recruited toward the lesional site, which in case of AD is the skin, nine chemokine ratios (Th2/Th1) were analyzed. After 4 months of dietary intervention, significantly lower CCL17/CXCL9 (MWU, *p*  \< 0.05), CCL20/CXCL9 (MWU, *p*  \< 0.05), and CCL22/CXCL9 (MWU, *p*  \< 0.01) ratios were observed in the active group as compared with the control group. Moreover, a reduction in CCL17/CXCL10 (W-test, *p* = 0.0781), CCL20/CXCL9 (W-test, *p*  \< 0.05), CCL20/CXCL10 (W-test, *p*  \< 0.05), and CCL20/CXCL11 (W-test, *p*  \< 0.01) ratios were observed over time within the active group. Interestingly, these ratios did not change in the control group over time (Figure [4](#) ). <figure> <p><img src="" /></p> <figcaption>Chemokine ratios calculated in the control (open dots, <em>n</em> = 20) and active (closed triangles, <em>n</em> = 21) groups before and after dietary intervention. CC chemokine ligand (CCL) 17/CXC chemokine ligand (CXCL) 9 <strong>(A)</strong> , CCL17/CXCL10 <strong>(B)</strong> , CCL22/CXCL9 <strong>(C)</strong> , CCL20/CXCL9 <strong>(D)</strong> , CCL20/CXCL10 <strong>(E)</strong> , and CCL20/CXCL11 <strong>(F)</strong> . Line represents median ± interquartile range. Significant differences within the groups are depicted with asterisks (non-paired W-test, * <em>p</em>  \< 0.05, \*\* <em>p</em>  \< 0.01) and between groups (using Mann–Whitney <em>U</em> -test, * <em>p</em>  \< 0.05, \*\* <em>p</em>  \< 0.01).</figcaption> </figure>


Extracted trial result:
Outcome: chemokines CXCL9 (continuous)
Intervention: extensively hydrolyzed whey-based formula with short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (9:1) and Bifidobacterium breve M-16V (active).
Comparator: extensively hydrolyzed whey-based formula without (control) short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (9:1) and Bifidobacterium breve M-16V (active).
Intervention group size: 13
Comparator group size: 13
Notes: mean and std not given, only median and interquartile range