Dendritic cells modification during sublingual immunotherapy in children with allergic symptoms to house dust mites

Federica Angelini, Valentina Pacciani, Stefania Corrente, Romina Silenzi, Alessandra Di Pede, Antonella Polito, Carla Riccardi, Silvia Di Cesare, Marie Laure Yammine, Paolo Rossi, Viviana Moschese, Loredana Chini

Rome, Italy

Author Affiliations: Division of Pediatrics, Tor Vergata University, Rome, Italy (Angelini F, Corrente S, Silenzi R, Polito A, Di Cesare S, Yammine ML, Rossi P, Moschese V, Chini L); DPUO, Bambino Gesù Children's Hospital, Rome, Italy (Angelini F, Pacciani V, Rossi P); Division of Neonatology, Bambino Gesù Children's Hospital, Rome, Italy (Alessandra Di Pede); Division of Pediatric Medicine, Bambino Gesù Children's Hospital, Rome, Italy (Riccardi C)

Corresponding Author: Federica Angelini, MD, PhD, University Department of Pediatrics (DPUO), Bambino Gesù Children's Hospital, Piazza S.Onofrio 4, 00165 Roma, Italy (Tel: +39 (06) 72596823; Fax +39 (06) 72596822; Email: angelini@med.uniroma2.it)

Background: The importance of dendritic cells (DCs) in the initiation of the Th2-mediated inflammatory response to allergens is well known and more recently it has been proposed that DCs have a pivotal role in maintaining tolerance to allergens. The aim of this study was to investigate whether the success of sublingual immunotherapy (SLIT) in allergic asthma is mediated by the induction of changes of DCs functions.

Methods: Ten children with allergic asthma sensitive to house dust mite were studied before and after 12 months of SLIT. Immature DCs were derived from peripheral blood monocytes cultured for 6 days in presence of interleukin (IL)-4 and GM-CSF and stimulated with lipopolysaccharide for the last 24 hours to induce maturation.

Results: After 12 months of SLIT, mature DCs derived from SLIT-treated patients showed a statistically significant defect of CD86 up-regulation, an increase of IL-10, and a reduction of IL-12 production.

Conclusion: SLIT induces changes in DCs functions that might be responsible for an impairment of T cell activation or drive T cells towards a regulatory activity, thus restoring immune tolerance to allergens.

Key words: allergy; CD86; dendritic cells; IL-10; sublingual immunotherapy

                   World J Pediatr 2017;7(1):24-30

Introduction

Despite this clinical evidence, its mode of action has not yet been fully elucidated. In the last years, some studies indicated that the mechanism of immunotherapy might be based on the increase of number and activity of regulatory T cells. Regulatory T cells appear to control the development of autoimmune diseases and transplant rejection and may also play a critical role in controlling asthma and allergy. In particular, type 1 regulatory T cells (Tr1) are a subset of CD4⁺ T cells that produce high levels of IL-10, low levels of IL-2 and no IL-4.^[2] It has been reported that they consistently represent the dominant subset specific for common environmental allergens in healthy individuals in contrast to the allergen-specific IL-4-secreting T cells frequently found in allergic individuals.^[3,4] Specific immunotherapy, by modifying Tr1 compartment, would then restore the natural immune tolerance to harmless antigens such as aeroallergens.^[5-7]

Accumulating evidences suggest that the generation of T regulatory cells in periphery is orchestrated by a particular subset of dendritic cells (DCs), whose phenotype and modality of function are topics of intense study. Early reports showed that repeated stimulation of naïve CD4 T cells with allogenic immature DCs induce Tr1 cells.^[8,9] Subsequently, it has been demonstrated that a specialized subset of DCs, termed tolerogenic DCs, can prime T cells to differentiate in Tr1 cells, thus promoting tolerance rather than immunity^[10] and several protocols have been developed to generate Tr1 cells in vitro by using tolerogenic DCs.^[11]

For years the gold standard of SIT was represented by the subcutaneous immunotherapy (SCIT), but the risk of severe systemic side effects and the need for frequent injections has limited the application of SCIT, especially in children. Nowadays, other kinds of immunotherapy such as sublingual-swallow immunotherapy (SLIT) may represent a valuable alternative to SCIT. SLIT has been shown to be safe and effective in several double-blind placebo-controlled studies in children affected by asthma and rhinitis.^[12-14] In 2001, the allergic rhinitis and its impact on asthma (ARIA) extended the indications of SLIT also to children and a recent meta-analysis concluded that SLIT significantly reduces both symptoms and the requirement of medications.^[15]

The immunological mechanism that underlies SLIT has only started to be investigated. It is conceivable that the sublingual administration route might induce immunological tolerance towards allergens involving cells and mediators specific of oral and intestinal mucosa. In particular, although it partially shares the same mechanisms of SCIT, it is also able to act on Langerhans cells present in oral mucosa. It seems to induce the secretion of allergen-specific IgG,^[16] to increase IL-10 and TGF-β expression and to enhance Tr1 functions.^[17,18]

The purpose of this study was to address the question whether SLIT promotes tolerogenic functions of DCs and whether it is possible to identify DCs specific biomarkers that may predict the success of SLIT in allergic patients.

Methods

Patients

We recruited 10 children (6 males and 4 females), aged 6.3 years on average (range: 4.5-9.2 years), affected by intermittent asthma and/or rhinitis, due to house dust mites (HDM) allergy. All children had positive skin prick test and/or specific IgE for Dermatophagoides Pteronyssinus (Dt.Pt.) and/or Dermatophagoides Farinae (Dt.F.). At the beginning and at the end of the study we assessed clinical score evaluating nasal symptoms (sneezing, itching nose, watery running and nasal blockage) and asthma daily and nocturnal symptoms (wheeze/breathless and dry cough during the night) using 0-4 scales (0=none, 1=mild intermittent, 2=mild persistent, 3=moderate persistent, 4=severe persistent).^[19,20] Patients' characteristics are detailed in Table 1. At the time of the enrollment all patients started SLIT (SARM, Rome, Italy), consisting the sublingual administration of allergen extract (HDM maximum dose 6000 BU/monthly).

The Ethical Committee of the Hospital approved the study and informed consent was obtained from every study participant after the nature of the study was explained.

Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from the patients, before HDM-SLIT (T0) and after 12 months (T12), by Ficoll density gradient centrifugation.

Generation of DCs

Immature DCs (iDCs) were generated from peripheral blood monocyte precursors. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by positive selection, using anti-CD14-conjugated magnetic Microbeads (MACS, Miltenyi Biotec, Belgish Gladbach, Germany) routinely resulting in >95% purity of the CD14⁺ population, as assessed by flow cytometry. DCs were derived from CD14⁺ cells cultured for 6 days in RPMI-1640 (Sigma, Milano, Italy) containing 10% FCS (Gibco, Carlsbad, CA, USA) and supplemented with GM-CSF (50 ng/mL, Leucomax, Sandoz AG, Nürnberg, Germany) and IL-4 (10 ng/mL, Sigma-Aldrich, St Louis, MO, USA), at the concentration of 4 × 10⁴ cells/ml.

Myeloid DCs, defined as CD14^–, CD1a⁺, CD11c⁺ cells, were then analyzed or further stimulated with lipopolysaccharide (LPS) from E. Coli (1 μg/mL, Sigma Chemicals, St Louis, MO, USA) for 24 hours to induce maturation.

Analysis of DCs phenotype by flow cytometry

DCs were stained for 20 minutes on ice with monoclonal antibodies conjugated to FITC or PE, specific for CD14, CD1a, CD11c, HLA-DR, CD80, CD86, CD83, as well as with isotype controls (all from Pharmingen, San Diego, CA, USA). For each sample, a minimum of 1×10⁴ events were acquired with a flow cytometer (FACSCalibur, Beckton Dickinson, San Jose, CA, USA) interfaced with Cellquest software (Beckton Dickinson).

Cytokines production assay

IL-12p70 and IL-10 concentrations in iDCs and mature DCs (mDCs) culture supernatants were determined by enzyme-linked immunosorbent assay (Human IL-12 and IL-10 ELISA, Endogen, Woburn, MA, USA). The test was performed in duplicate according to the instructions of the manufacturer.

Statistical analysis

The SPSS for Windows 17.0 was used for statistical analysis. Experimental data were tested for distribution according to Kolmogorov-Smirnov, and all the surface cell markers were normally distributed. The differences between groups were therefore analyzed with Student's t test and a value of P<0.05 was considered statistically significant.

Results

Clinical outcome

During the observational period, none of the children experienced severe exacerbations of asthmatic symptoms necessitating either hospital admission or treatment with a short course of oral steroids, without viral or bacterial infections. As described in Table 1, after 12 months of treatment we observed an improvement of both nasal and asthma symptoms, as indicated by the reduction of the score. Concerning laboratory parameters, during SLIT treatment, we observed a significant modification only for specific IgE to Dt. F. (P= 0.038).

DCs phenotypes

We analyzed expressions of HLA-DR, CD80, CD86 and CD83 on DCs derived from SLIT-treated allergic asthmatic children (n=10) at T0 and T12 (Fig. 1). After 12 months of follow-up, no statistically significant changes were found in the expressions of HLA-DR, CD80 and CD83 on both iDC and mDC. A slight reduction of HLA-DR and CD80 expressions was detected (Table 2 and Fig. 2A).

Interestingly, the expression of CD86 was reduced in both iDCs and mDCs at T12 compared to T0, and the decrease was statistically significant for mDC (P= 0.00006) (Table 2 and Fig. 2A).

IL-10 and IL-12p70 production by DCs

Upon maturation with LPS, mDCs from allergic patients produced detectable amount of cytokines as determined in culture supernatants. IL-10 production was increased after 12 months of SLIT (373±521 vs. 276±281 pg/ml), while IL-12 production was decreased (141±257 vs. 1598±1614 pg/ml) (Fig. 2B).

In the supernatants of iDCs cultures of both IL-10 and IL-12p70 were undetectable.

Discussion

To elucidate the immunological changes induced by SLIT, we studied the function of DCs in asthmatic patients receiving SLIT for 12 months. We observed a statistically significant reduction over time of the expression of CD86, an important costimulatory molecule for T cells, on mDCs. Differently, CD80 expression, as well as HLA-DR expression, was substantially unchanged. CD86 appears indeed to be more important than CD80 for the induction of the allergic immune response.^[21,22] In murine models of allergic airway inflammation, blockade of CD86, but not of CD80, abolishes airway eosinophilia and airway hyperresponsiveness.^[23,24] To note, the defective up-regulation of CD86 on mDC at T12 was consistent with the increase of IL-10 that can be detected at the same time point. Indeed, IL-10 can cause a down-regulation of costimulatory molecules expression^[25] leading to an impairment of T cell-stimulatory ability of DCs, as well as to an induction of T anergy.^[26,27] Therefore, the reduction of CD86 expression that we observed during SLIT might have an important role in the induction of tolerance or in the suppression of immune response to allergens.

Moreover, we observed a contemporary decrease of IL-12 production by DCs at T12. This could be secondary to the increased secretion of IL-10 since it is known that IL-10 can downregulate the immune response and counteracts the activity of IL-12.^[28] Although the amounts of IL-10 produced by mDCs were quite variable among patients, here we provide the first evidence, to our knowledge, of an increased production of IL-10 by DCs during SLIT. This finding is supported by previous studies demonstrating that SLIT increases IL-10 production in PBMCs of allergic patients.^[29-32] In particular, in the study by Ciprandi et al,^[29] IL-10 production was induced by Dermatophagoides Farinae, but also by recall antigens and phytohemagglutinin, thereby raising the possibility that some IL-10 secreting cells were not T lymphocytes.

Our findings suggest that the immunological mechanism of SLIT might reside in the induction of IL-10 production by DCs. Some previous reports on cohorts of patients treated with SLIT described that an early increase of IL-10 mRNA positively correlates with an improvement of the clinical score.^[33,34] It has been recently shown that IL-10 can induce the differentiation of T cells with antigen-specific regulatory activity, directly or via induction of an IL-10 producing subset of DCs.^[34-37] Therefore, it is conceivable that an increase of IL-10 production by the DCs during the first months of SLIT might drive an expansion of the T regulatory cell population, which would be responsible for the clinical improvement usually observed after the first year of immunotherapy. This interpretation is supported by the recent work of Koya et al,^[38] who demonstrated that DCs treated with IL-10 produced, in turns, considerable amounts of IL-10, and that the adoptive transfer of IL-10-treated DCs leads to an increase of CD4⁺IL-10⁺T cells in the lung of sensitized recipient mice. Interestingly, the negative regulatory effects exhibited by the IL-10–treated DCs were dependent on endogenous production of IL-10 because DCs from IL-10–deficient mice, despite differentiation in the presence of IL-10, failed to exhibit the negative regulatory effects in any of these assays.^[38]

In summary, our results are consistent with the hypothesis that the induction of tolerance by SLIT involves a modification of DCs functions, although further studies on a larger group of patients might help to elucidate if IL-10 or CD86 is a reliable biomarker of the success of immune therapy. In the light of the recent research it appears that DCs have to manage two opposite tasks: on one hand they promote pro-inflammatory reactions and actively induce T-cell responses, on the other hand they have an important function as 'silencers' within the immune system by sending out anti-inflammatory, tolerance inducing signals. The last unique capacity of DCs has opened several exciting possibilities to exploit DCs for recovering tolerance to allergens and protecting from allergic reactions. The importance of therapies specifically targeting the immune system as the only disease-modifying treatment for allergic diseases is evident. In particular there is a great need for therapies aimed to prevent or to block the initial development of the allergic inflammation. The work discussed herein might help to define the role of DCs in the pathogenesis of allergy and to implement therapies attempting to intervene in the early phases of allergic inflammation.

Acknowledgements

We wish to thank Francesca Monzo for the excellent technical support and Andrea Pierantozzi for the statistical analysis of the data.

Funding: This work was supported by grants from the Ministero dell'Istruzione, Universita'e Ricerca (MIUR) and from the Bambino Gesù Children's Hospital of Rome, Italy.

Ethical approval: This study was approved by the Ethical Committee of the Hospital.

Competing interest: The authors declare no conflicts of interest.

Contributors: Angelini F had primary responsibility for protocol development, performed the final data analysis and the writing of the manuscript. Pacciani V performed most of the experiments. Di Cesare S performed part of the flow cytometry experiments. Corrente S, Silenzi R, Di Pede A, Polito A, Riccardi C, and Yammine ML performed patient screening, enrollment and outcome assessment. Rossi P supervised laboratory activities. Moschese V participated in the development of the protocol and patient screening. Chini L had primary responsibility for protocol development, supervised the design and execution of the study and contributed to the writing of the manuscript.

References

1   Bousquet J, Lockey R, Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper. J Allergy Clin Immunol 1998;102:558-562.

2   Akdis CA, Akdis M. Mechanisms and treatment of allergic disease in the big picture of regulatory T cells. J Allergy Clin Immunol 2009;123:735-746; quiz 747-748.

3   Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, et al. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med 2004;199:1567-1575.

4   Ozdemir C, Akdis M, Akdis CA. T regulatory cells and their counterparts: masters of immune regulation. Clin Exp Allergy 2009;39:626-639.

5   Francis JN, Till SJ, Durham SR. Induction of IL-10⁺CD4⁺CD25⁺ T cells by grass pollen immunotherapy. J Allergy Clin Immunol 2003;111:1255-1261.

6   Gardner LM, Thien FC, Douglass JA, Rolland JM, O'Hehir RE. Induction of T 'regulatory' cells by standardized house dust mite immunotherapy: an increase in CD4⁺ CD25⁺ interleukin-10⁺ T cells expressing peripheral tissue trafficking markers. Clin Exp Allergy 2004;34:1209-1219.

7   Jutel M, Akdis CA. T-cell regulatory mechanisms in specific immunotherapy. Chem Immunol Allergy 2008;94:158-177.

8   Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 2000; 192:1213-1222.

9   Nguyen KD, Vanichsarn C, Nadeau KC. Impaired IL-10-dependent induction of tolerogenic dendritic cells by CD4+CD25hiCD127lo/- natural regulatory T cells in human allergic asthma. Am J Respir Crit Care Med 2009;180:823-833.

10 Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine modulation comes of age. Blood 2006;108:1435-1440.

11 Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 2006;212:28-50.

12 Bousquet J, Scheinmann P, Guinnepain MT, Perrin-Fayolle M, Sauvaget J, Tonnel AB, et al. Sublingual-swallow immunotherapy (SLIT) in patients with asthma due to house-dust mites: a double-blind, placebo-controlled study. Allergy 1999;54:249-260.

13 La Rosa M, Ranno C, André C, Carat F, Tosca MA, Canonica GW. Double-blind placebo-controlled evaluation of sublingual-swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 1999;104:425-432.

14 Bousquet J, Demoly P. Specific immunotherapy―an optimistic future. Allergy 2006;61:1155-1158.

15 Wilson DR, Lima MT, Durham SR. Sublingual immunotherapy for allergic rhinitis: systematic review and meta-analysis. Allergy 2005;60:4-12.

16 Moingeon P, Batard T, Fadel R, Frati F, Sieber J, Van Overtvelt L. Immune mechanisms of allergen-specific sublingual immunotherapy. Allergy 2006;61:151-165.

17 Bohle B, Kinaciyan T, Gerstmayr M, Radakovics A, Jahn-Schmid B, Ebner C. Sublingual immunotherapy induces IL-10-producing T regulatory cells, allergen-specific T-cell tolerance, and immune deviation. J Allergy Clin Immunol 2007;120:707-713.

18 O'Hehir RE, Gardner LM, de Leon MP, Hales BJ, Biondo M, Douglass JA, et al. House dust mite sublingual immunotherapy: the role for transforming growth factor-beta and functional regulatory T cells. Am J Respir Crit Care Med 2009;180:936-947.

19 Demoly P, Allaert FA, Lecasble M, Bousquet J; PRAGMA. Validation of the classification of ARIA (allergic rhinitis and its impact on asthma). Allergy 2003;58:672-675.

20 LeBlanc A, Robichaud P, Lacasse Y, Boulet LP. Quantification of asthma control: validation of the Asthma Control Scoring System. Allergy 2007;62:120-125.

21 Tsuyuki S, Tsuyuki J, Einsle K, Kopf M, Coyle AJ. Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness. J Exp Med 1997;185:1671-1679.

22 Larché M, Till SJ, Haselden BM, North J, Barkans J, Corrigan CJ, et al. Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics. J Immunol 1998;161:6375-6382.

23 Haczku A, Takeda K, Redai I, Hamelmann E, Cieslewicz G, Joetham A, et al. Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice. Am J Respir Crit Care Med 1999;159:1638-1643.

24 Pereira C, Tavares B, Loureiro G, Paiva A, Henriques A, Abrantes M, et al. Dynamics of CD86 expression on allergic inflammation―new insights. Recent Pat Inflamm Allergy Drug Discov 2009;3:128-131.

25 Creery WD, Diaz-Mitoma F, Filion L, Kumar A. Differential modulation of B7-1 and B7-2 isoform expression on human monocytes by cytokines which influence the development of T helper cell phenotype. Eur J Immunol 1996;26:1273-1277.

26 Steinbrink K, Jonuleit H, Müller G, Schuler G, Knop J, Enk AH. Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood 1999;93:1634-1642.

27 Sato K, Yamashita N, Matsuyama T. Human peripheral blood monocyte-derived interleukin-10-induced semi-mature dendritic cells induce anergic CD4(+) and CD8(+) T cells via presentation of the internalized soluble antigen and cross-presentation of the phagocytosed necrotic cellular fragments. Cell Immunol 2002;215:186-194.

28 Romagnani S. Biology of human TH1 and TH2 cells. J Clin Immunol 1995;15:121-129.

29 Ciprandi G, Fenoglio D, Cirillo I, Vizzaccaro A, Ferrera A, Tosca MA, et al. Induction of interleukin 10 by sublingual immunotherapy for house dust mites: a preliminary report. Ann Allergy Asthma Immunol 2005;95:38-44.

30 Savolainen J, Jacobsen L, Valovirta E. Sublingual immunotherapy in children modulates allergen-induced in vitro expression of cytokine mRNA in PBMC. Allergy 2006;61:1184-1190.

31 Cosmi L, Santarlasci V, Angeli R, Liotta F, Maggi L, Frosali F, et al. Sublingual immunotherapy with Dermatophagoides monomeric allergoid down-regulates allergen-specific immunoglobulin E and increases both interferon-gamma- and interleukin-10-production. Clin Exp Allergy 2006;36:261-272.

32 Tsai YG, Chiou YL, Chien JW, Wu HP, Lin CY. Induction of IL-10⁺ CD4⁺ CD25⁺ regulatory T cells with decreased NF-kappaB expression during immunotherapy. Pediatr Allergy Immunol 2010;21:e166-e173.

33 Savolainen J, Laaksonen K, Rantio-Lehtimäki A, Terho EO. Increased expression of allergen-induced in vitro interleukin-10 and interleukin-18 mRNA in peripheral blood mononuclear cells of allergic rhinitis patients after specific immunotherapy. Clin Exp Allergy 2004;34:413-419.

34 Ciprandi G, Cirillo I, Fenoglio D, Marseglia G, Tosca MA. Sublingual immunotherapy induces spirometric improvement associated with IL-10 production: preliminary reports. Int Immunopharmacol 2006;6:1370-1373.

35 Steinbrink K, Graulich E, Kubsch S, Knop J, Enk AH. CD4(+) and CD8(+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 2002;99:2468-2476.

36 Sato K, Yamashita N, Baba M, Matsuyama T. Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood 2003;101:3581-3589.

37 Wakkach A, Fournier N, Brun V, Breittmayer JP, Cottrez F, Groux H. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003;18:605-617.

38 Koya T, Matsuda H, Takeda K, Matsubara S, Miyahara N, Balhorn A, et al. IL-10-treated dendritic cells decrease airway hyperresponsiveness and airway inflammation in mice. J Allergy Clin Immunol 2007;119:1241-1250.

Received September 21, 2009 Accepted after revision May 19, 2010