Cytokine profile in children with food allergy following liver transplantation

LTX in children is associated with increased risk of food allergy, and the mechanisms underlying this are unknown. We wanted to study whether plasma cytokine profile differed in liver transplanted children, with and without food allergy, and whether it differed from untransplanted children with CLD.


| INTRODUC TI ON
LTX can be lifesaving in children, but is also associated with complications. Several studies have shown that food allergy is common in children after LTX, many of them with severe allergic reactions. [1][2][3][4][5][6] The prevalence of new-onset food allergy in liver transplanted children has been reported to be 4%-40.5% [6][7][8][9][10] and is accompanied by an increased risk of eczema and asthma. 6 The majority of patients with food allergy are young, and the risk of developing food allergy is highest the first year after LTX. 11 The mechanisms are unknown. Food allergy is an inappropriate response of the immune system triggered by the ingestion of a food protein allergen. 12 Cytokines produced by a number of cell types regulate different functions including innate immunity, acquired immunity, and inflammatory responses. In food allergy, the immune response is biased toward a type 2 cytokine-associated phenotype. 13  Furthermore, a change in immunosuppression from tacrolimus to another immunosuppressive drug reduces the risk of allergy. 4 Tacrolimus is not the only factor as food allergy is rarely seen after kidney transplantation. 10,16,17 One study found a significant higher number of natural killer (NK) cells in liver transplanted children compared with kidney transplanted children and hypothesized that NK cells might be involved in the mechanism. 18 Nahum et al 19 studied stimulated leukocytes in post-liver transplant children with food allergy compared with non-allergic transplant patients and found increased levels of IL-5, but decreased levels of IL-10. We aimed to investigate whether there is a persistent change in the cytokine profile in liver transplanted children with and without food allergy, and also to investigate whether there is a difference in cytokine profile between the liver transplanted children and the children with CLD who have not been transplanted.

| MATERIAL S AND ME THODS
The study was approved by the Institutional Review Board at Oslo University Hospital and by the Regional Ethics Committee (08/324d, 2008/6203). Written informed consent was obtained from patients or parents.

| Study design and population
The study was designed as a case-control study with liver transplanted patients with (n = 9) and without (n = 13) reported food allergy, and with a group of children with CLD (n = 7) for comparison. Twenty-two patients who underwent orthotopic LTX at Oslo University Hospital during the period from 1995 to 2009 were included. All patients received a split liver from deceased donors. The immunosuppression protocol currently used consists of tacrolimus, mycophenolic acid, and prednisolone in tapering doses. A history of allergic reactions was recorded using a questionnaire. The type of immunosuppressive therapy was recorded.

| Allergy
Total IgE, serum eosinophilic cationic protein (ECP), and allergenspecific IgE were quantified using the Phadia(R)/Thermo Fisher Scientific ImmunoCAP (Phadia) system. The panels included specific IgE for cow's milk, egg, peanut, soybean, wheat, cod, horse, cat, grass, birch, mugwort (common wormwood), mites, molds (cladosporium and alternaria). Sensitization was defined as specific IgE levels ≥0.35 kU/L Reported food allergy was defined as a reported history of symptoms like pruritus, tongue-, face-, or lip-edema, dyspnea or hoarseness, urticaria or anaphylaxis immediately after exposure to a food suspected to be linked to the reaction. Eczema was defined as reported use of treatment of eczema prescribed by a doctor. Asthma was defined as the patient using asthma medication (during the last 12 months) prescribed by a doctor because of asthma.

| Cytokines
Plasma samples were obtained together with routine sampling at the outpatient clinic. Samples were centrifuged and stored at −70°C until analysis. Plasma was diluted 1:3 in Bio-plex sample diluent before analysis. Cytokines were measured using enzyme-linked immu-

| Statistical analysis
Data are reported as mean and standard deviation or median and interquartile range. Statistical analyses were performed using Mann-Whitney U test for independent samples. A two-sided P value less than .05 was considered statistically significant.

| RE SULTS
Twenty-two children who had previously undergone LTX (nine children with food allergy and 13 without food allergy) and seven untransplanted children with CLD were included in the study.
Diagnoses are given in Table 1. Median age at transplantation was 0.7 (0.6-7.5) years, and the median time from transplantation to allergy testing was 7.6 (0.5-8.9) years. Age at testing was not different

| Transplanted children and food allergy
Nine out of 22 transplanted patients had reported food allergy. The reported allergens were egg, kiwi, cod, nuts, caviar, chicken, turkey, salami, shrimps, coconut, chocolate, banana, tomato, cheese, and milk. All nine patients with reported allergy had sensitization to food allergens, and one of the patients without reported food allergy had sensitization to food allergens ( Table 2). Sensitizations to aeroallergens (grass, birch, mugwort) occurred in four patients in the reported food allergy group and in two patients in the transplanted children without reported food allergy.
The median age at transplantation was significantly lower in the food allergy group compared to the transplanted patients without food allergy. The median age at testing for the transplanted children with and without food allergy was 8.6 (4.1-10.5) years and 12.8 (5.8-15.4) years, respectively. The median total IgE was higher in transplanted children with food allergy (581 [103-2166] IU/L) than transplanted without food allergy (13   (P = .032), respectively. All of the patients with food allergy were sensitized to one or more food allergens.

| Transplanted children with and without tacrolimus
Tacrolimus is associated with food allergy in liver transplanted children. In these selected transplanted patients, 17 were currently

| Transplanted children with and without tacrolimus and with and without food allergy
There was no significant difference in the cytokines in the transplan-

| Patients with epinephrine
Six out of the nine children with reported food allergy had been

| Clinical and biochemical effects of changing immunosuppression
Two patients had severe food allergy and had to be converted from tacrolimus-based immunosuppression to MMF and corticosteroids.
Patient 1 was a 3-year-old boy. The preconversion blood test was taken 6 days before conversion, and the post-conversion blood test was taken 2 months after conversion. The patient was liver trans-   the CLD group. The other factor is that allergy and asthma are associated with high MIP-1b [23][24][25] and even though these patients were treated with tacrolimus, MIP-1b was elevated because of allergy. MIP-1b is a pro-inflammatory cytokine involved in immune responses toward inflammation and infection. 26 Change in immunosuppression may affect both factors. The levels of MIP-1b increased in patient 1 after conversion, but decreased in patient 2.

| D ISCUSS I ON
Of note, the time frame in the two patients was different as the blood test was taken 2.5 months following conversion in patient 1, but after 4 years in patient 2. Our hypothesis is that MIP-1b decreases after some time following conversion when the allergy effect disappears.
Use of tacrolimus in liver transplanted children has been linked to de novo food allergy. Tacrolimus causes a shift toward a Th2 cytokine profile by inhibiting IL-2. 2, 19 We did not however find a significant difference in the cytokine profile in the liver transplanted children treated with tacrolimus compared with those treated with cyclosporine or MMF. This may be due to a small number of patients in these subgroups. Tacrolimus is not the only factor as food allergy is rarely seen after kidney transplantation. 16 PDGF is a growth factor highly involved in the development of liver fibrosis and is associated with chronic inflammation. 30,31 Chemokines (as RANTES or chemokine ligand 5) are relevant in allergy because of their role in regulating leukocyte recruitment, but also for cellular activation, inflammatory mediator release, promotion of Th2 inflammatory responses, and regulation of IgE. 32 RANTES has been linked to recruitment of Th2 cells, 33 and TNFα is thought to play a major role in the pathogenesis of allergy and inflammatory diseases as it is produced at an early stage of allergen sensitization and continues to promote the inflammation cascade throughout the allergy development. 34 The clinical improvement in food allergy, the reduction of total and specific IgE, and the change in cytokine profile support that tacrolimus plays a significant role in the development of transplant-acquired food allergy as reported by several other studies. [2][3][4]7,15 Few studies, to our knowledge, have investigated the plasma cytokine profile after LTX in children. [35][36][37] Ganschow et al demonstrated that a tendency toward a Th2 cytokine profile corresponded with a better graft survival in infants after LTX 35 and early cytokine measurements after LTX showed lower levels of Th2 cytokines in patients with graft rejection. 36,37 In the current study, we found that there was a significant difference in the cytokine profile between liver transplanted children and children with CLD. The transplantation group had higher levels of IL-1b, IL-5, IL-7, IL-13, GCSF, IFNγ, and MIP-1a compared with the CLD group, but lower levels of MIP-1b.
Th2-driven inflammation is associated with increased levels of IL-4, This could give valuable insight into a particular phenotype more prone to develop food allergy than others making it possible to tailor a more personalized immunosuppression regimen. Further studies are needed to assess this.

ACK N OWLED G M ENTS
The authors are grateful for the technical assistance of Monica Atneosen-Åsegg.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.