Clinical efficacy of sublingual immunotherapy is associated with restoration of steady-state serum lipocalin 2 after SLIT: a pilot study

Franziska Roth-Walter, René Schmutz, Nadine Mothes-Luksch, Patrick Lemell, Petra Zieglmayer, René Zieglmayer, Erika Jensen-Jarolim, Franziska Roth-Walter, René Schmutz, Nadine Mothes-Luksch, Patrick Lemell, Petra Zieglmayer, René Zieglmayer, Erika Jensen-Jarolim

Abstract

Background: So far, only a few biomarkers in allergen immunotherapy exist that are associated with a clinical benefit. We thus investigated in a pilot study whether innate molecules such as the molecule lipocalin-2 (LCN2), with implications in immune tolerance demonstrated in other fields, may discriminate A) between allergic and non-allergic individuals, and B) between patients clinically responding or non-responding to sublingual allergen immunotherapy (SLIT) with house dust mite (HDM) extract. Moreover, we assessed haematological changes potentially correlating with allergic symptoms.

Methods: LCN2-concentrations were assessed in sera of healthy and allergic subjects (n = 126) as well as of house dust mite (HDM) allergics before and during HDM- sublingual immunotherapy (SLIT) in a randomized, double-blind, placebo-controlled trial for 24 weeks. Sera pre-SLIT (week 0), post-SLIT (week 24) and 9 months after SLIT were assessed for LCN2 levels and correlated with total nasal symptom scores (TNSS) obtained during chamber challenge at week 24 in patients receiving HDM- (n = 31) or placebo-SLIT (n = 10).

Results: Allergic individuals had significantly (p < 0.0001) lower LCN2-levels than healthy controls. HDM-allergic patients who received HDM-SLIT showed a significant increase in LCN2 9 months after termination of HDM-SLIT (p < 0.001), whereas in subjects receiving placebo no increase in LCN2 was observed. Among blood parameters a lower absolute rise in the lymphocyte population (p < 0.05) negatively correlated with symptom improvement (Pearson r 0.3395), and a lower relative increase in the neutrophils were associated with improvement in TNSS (p < 0.05). LCN2 levels 9 months after immunotherapy showed a low positive correlation with the relative improvement of symptoms (Pearson r 0.3293). LCN2-levels 9 months off-SLIT were significantly higher in patients whose symptoms improved during chamber challenge than in those whose symptoms aggravated (p < 0.01).

Conclusion: Serum LCN2 concentrations 9 months off-SLIT correlated with clinical reactivity in allergic patients. An increase in the LCN2 levels 9 months after HDM-SLIT was associated with a clinical benefit. Serum LCN2 may thus contribute to assess clinical reactivity in allergic patients.

Trial registration: Part of the data were generated from clinicaltrials.gov Identifier NCT01644617.

Keywords: Allergen; Clinical efficacy; Innate; Lipocalin 2; Sublingual immunotherapy.

Conflict of interest statement

Samples were obtained from two distinct patient cohorts. Serum samples of allergic and non-allergic individuals were collected in collaboration with the allergy diagnosis and study center AllergyCare, Vienna, Austria. Approval for the retrospective analysis was obtained from the ethics committee of the Medical University of Vienna and conducted in accordance with the Helsinki Declaration of 1975. Immunotherapy samples from the double-blind placebo-controlled trial NCT01644617 were obtained from Vienna Challenge Chamber. Details of efficacy and safety outcome of this trial are described elsewhere [29, 30]. The study was conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. Written informed consent was obtained from participants before the study, and the protocol was approved by an independent ethics committee.All authors have seen and approved the last version.EJJ and FRW are inventors of EP2894478, owned by Biomedical International R + D GmbH, Vienna, Austria. The other authors declare no conflicts of interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Decreased serum LCN2 levels in allergic compared to non-allergic subjects. Serum LCN2 concentrations were assessed in (a) allergic (n = 63) and non-allergic (n = 46) individuals and (b) assessed by gender. c Within the allergic cohort, allergic women had lower LCN2-levels than allergic men, whereas no gender-disparity was observed in the non-allergic cohort. Statistical analyses were conducted with Student’s t-test. *p < 0.05, ***p < 0.001, ****p < 0.0001
Fig. 2
Fig. 2
Immunological changes of subjects undergoing sublingual immunotherapy, SLIT. Individual changes in the (a) absolute lymphocyte and relative neutrophil counts in active or placebo-treated participants, (b) in the relative lymphocyte and neutrophil counts of individuals benefitting or not from sublingual treatment according to TNSS. Statistical analyses were conducted with Student’s t-test
Fig. 3
Fig. 3
Correlation of relative total nasal symptom score (TNSS) improvement with changes in (a) absolute lymphocyte, (b) relative lymphocyte numbers as well as changes of (c) absolute and (d) relative neutrophil counts before and after 6 months treatment. Correlation were obtained using Pearson’s rank method
Fig. 4
Fig. 4
LCN2 serum concentrations rise in the active, but not placebo-treated group approximately 9 months after end of sublingual treatment. LCN2-concentration and total nasal symptom scores (TNSS) in (a) active and (b) placebo-treated participants. Statistical analyses were conducted using one-way ANOVA using Tukey’s multiple comparisons test for post-hoc analyses. **** p < 0.0001, *** p < 0.001
Fig. 5
Fig. 5
LCN2 concentrations 9 months off-SLIT are higher in subjects whose symptoms ameliorated. (a) LCN2-concentration after end of treatment and (b) individual relative improvement of the total nasal symptom score, TNSS, in the active and placebo-treated groups. The threshold of 20% for amelioration of symptoms (grey area) were set to group study participants according to their clinical outcome. (c) LCN2-concentrations 9 months off-SLIT of patients according to their clinical outcome, irrespective whether the subjects belonged to the placebo- or active treated group. (d) Correlation of absolute LCN2-levels 9 month off-SLIT with subjects’ relative improvement in the TNSS. Statistical analyses were conducted using one-way ANOVA using Tukey’s multiple comparisons test for post-hoc analyses. Correlation was obtained using Pearson’s rank method
Fig. 6
Fig. 6
LCN2 correlate with neutrophils. Correlation of natural log-transformed LCN2 concentration occurring 9 months off-SLIT with changes in the (a) absolute and (b) relative numbers of the neutrophil population. Correlation was obtained using Pearson’s rank method

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