Allergy Asthma Immunol Res.  2018 Jul;10(4):406-419. 10.4168/aair.2018.10.4.406.

Tolerogenic Dendritic Cells Reduce Airway Inflammation in a Model of Dust Mite Triggered Allergic Inflammation

• Aragão-França LS
• Rocha VC
• Cronemberger-Andrade A
• Costa FH
• Vasconcelos JF
• Athanazio
• Silva DN
• Santos ES
• Meira CS
• Araújo CF
• Cerqueira
• Cardillo F
• Alcântara-Neves NM
• Soares MB
• Pontes-de-Carvalho LC

• KMID: 2421675
• DOI: http://doi.org/10.4168/aair.2018.10.4.406

Abstract

PURPOSE
The use of tolerogenic dendritic cells (TolDCs) to control exacerbated immune responses may be a prophylactic and therapeutic option for application in autoimmune and allergic conditions. The objective of this work was to evaluate the effects of TolDC administration in a mouse model of allergic airway inflammation caused by mite extract.
METHODS
Mouse bone marrow-derived TolDCs were induced by incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and dexamethasone, and then characterized by flow cytometry and cytokine production by enzyme-linked immunosorbent assay (ELISA). For the in vivo model of Blomia tropicalis-induced allergy, mice transplanted with antigen-pulsed TolDCs were sensitized intraperitoneally with B. tropicalis mite extract (BtE) adsorbed to aluminium hydroxide. After challenge by nasal administration of BtE, bronchoalveolar lavage fluid (BALF), lungs, spleen and serum were collected for analysis.
RESULTS
Induction of TolDCs was efficiently achieved as shown by low expression of major histocompatibility complex (MHC) II, programmed death-ligand (PD-L) 2 and pro-inflammatory cytokine production, and up-regulation of interleukin (IL)-10, upon LPS stimulation in vitro. Transplantation of 1 or 2 doses of BtE-pulsed TolDCs reduced the number of inflammatory cells in BALF and lungs as well as mucus deposition. Moreover, compared to saline-injected controls, TolDC-treated mice showed lower serum levels of anti-BtE immunoglobulin E (IgE) antibodies as well as reduced Gata3 and IL-4 gene expression in the lungs and decreased IFN-γ levels in the supernatant of splenocyte cultures Transplantation of TolDCs increased the percentage of the regulatory T cells in the spleen and the lungs.
CONCLUSIONS
Preventive treatment with TolDCs protects against dust mite-induced allergy in a mouse model, reinforcing the use of tolerogenic dendritic cells for the management of allergic conditions.

Keyword

Dendritic cells; asthma; allergens, house dust mites; immunotherapy and tolerance induction

MeSH Terms

Administration, Intranasal
Animals
Antibodies
Antigens, Dermatophagoides
Asthma
Bronchoalveolar Lavage Fluid
Dendritic Cells*
Dexamethasone
Dust*
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Gene Expression
Granulocyte-Macrophage Colony-Stimulating Factor
Hypersensitivity
Immunoglobulin E
Immunoglobulins
In Vitro Techniques
Inflammation*
Interleukin-4
Interleukins
Lung
Major Histocompatibility Complex
Mice
Mites*
Mucus
Spleen
T-Lymphocytes, Regulatory
Up-Regulation
Antibodies
Antigens, Dermatophagoides
Dexamethasone
Dust
Granulocyte-Macrophage Colony-Stimulating Factor
Immunoglobulin E
Immunoglobulins
Interleukin-4
Interleukins

Figure

• 

  Fig. 1 Immunophenotype of DCs differentiated from bone marrow cells in the presence of dexamethasone. (A) Protocol for the generation of tolerogenic DCs from A strain mouse bone marrow. Control cells were cultured in differentiation medium and stimulated with LPS in the absence of dexamethasone. (B–H) Cells were stained with antibodies against surface markers as indicated. Debris and dead cells were excluded on the basis of forward-scatter and side-scatter. (B) Representative dot plots depicting the percentages of CD11c+CD11b+ cells. (C-H) Histograms of CD40 (C), CD80 (D), CD86 (E), PD-L1 (F), PD-L2 (G), and MHC II (H) cells gated on CD11c+CD11b+ double positive cells. DCs are represented by filled gray histograms and TolDCs by black lines; isotype controls are shown by dotted gray lines. Results are representative of 3 independent experiments. DC, dendritic cell; LPS, lipopolysaccharide; PD-L, programmed death-ligand; MHC, major histocompatibility complex; TolDC, tolerogenic dendritic cell.

• 

  Fig. 2 Anti-inflammatory cytokine production profile of DCs differentiated from bone marrow cells in the presence of dexamethasone. Control cells were left to differentiate in the absence of dexamethasone. The supernatants from cultures were harvested 24 hours after activation with lipopolysaccharide for TNF-α (A), IL-12p70 (B), IL-1β (C) and MCP-1 (D), and the measurement of IL-10 (E) was performed 48 hours after stimuli with LPS. Cytokines were quantified by sandwich ELISA. The data represented is the median with a range of 4 (TNF-α, IL-12p70 and IL-1β), 7 (MCP-1) and 3 (IL-10), independent of experiments. DC, dendritic cell; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay. *P<0.05 (Mann-Whitney's U test).

• 

  Fig. 3 Evaluation of Th2 parameters by dexamethasone-induced tolerogenic DCs. Experimental protocol of TolDC therapy in a mouse model of allergy to B. tropicalis (A). Mice were pre-treated with 1 or 2 doses of TolDCs prior to sensitization with BtE. Mice were euthanized 24 hours after the last challenge with BtE. The cellularity in BALF from naïve or Blomia-challenged mice, treated with 1 (TolDCs 1×) or 2 (TolDCs 2×) doses of TolDCs was evaluated. Total cell number (B), number of eosinophils in 200 cells (C) and differential (D) in BALF. Values are expressed as mean±SEM of 5–8 mice per group, in 1 of the 5 experiments performed. Th2, T helper type 2; DC, dendritic cell; TolDC, tolerogenic dendritic cell; BtE, B. tropicalis extract; BALF, bronchoalveolar lavage fluid; SEM, standard error of the mean. *P<0.05; †P<0.01; ‡P<0.001; and §P<0.05 compared to the naïve and TolDC 2x groups (B and C, Dunn's multiple comparison test; D, Newman-Keuls multiple comparison test).

• 

  Fig. 4 Immunomodulation by TolDCs. Serum levels of total IgE (a) and BtE-specific IgE (B) were measured by ELISA. Relative expression to Gapdh of Gata3 (C) and IL-4 (D) of lung fragments of A strain mice treated with TolDCs previously to allergy induction, by Real-Time PCR. Levels of IFN-γ in the supernatant of splenocyte cultures were evaluated using CBA assay (E). Values are expressed as mean±SEM of 5–8 mice per group, in 1 of the 5 experiments performed. TolDC, tolerogenic dendritic cell; BtE, B. tropicalis mite extract; IgE, immunoglobulin E; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; IFN, interferon; CBA, cytometric bead array; SEM, standard error of the mean. *P<0.05; †P<0.01; ‡P<0.001 (Newman-Keuls multiple comparison test).

• 

  Fig. 5 Airway inflammation of TolDC-treated mice. The right lobe sections of the lungs were stained with H&E for the quantification of inflammatory cells by optical microscopy. For each of the lung 10 fields (400×) were analyzed per section, and the data used to calculate the mean number of cells per mm2. Scale bar, 50 µm. (A–D) Representative hematoxylin and eosin-stained sections of the lungs of A strain mice. (A) Normal tissue of an untreated animal. (B) Inflammatory infiltrate with a predominance of polymorphonuclear around bronchi and arterioles in an animal from the positive control group (Blomia). (C and D) Reduced lung perivascular infiltration by polymorphonuclear cells, in a Blomia-sensitized animal pre-treated with TolDCs 1× and TolDCs 2×, respectively. (E) Infiltration of inflammatory cells per mm2. (F) Relative expression to Gapdh of Ptprc. The data is representative of 3 independent experiments. TolDC, tolerogenic dendritic cell. *P<0.05; †P<0.01; ‡P<0.001 (Newman-Keuls multiple comparison test).

• 

  Fig. 6 Mucus analysis of lungs from allergic and tolerized mice. Lung sections of (A) control and (B) allergic mice treated with vehicle, (C) tolerized with TolDCs 1× or (D) tolerized with TolDCs 2×. Narrow arrows indicate areas of alcian blue+ cells (original magnification ×400). Scale bar, 50 µm. (E) Quantification of mucus production on alcian blue-stained lung sections. The area of alcian blue staining was estimated by morphometric analysis. Data is expressed as means±SEM of 5–8 mice per group, in 1 of the 2 experiments performed. TolDC, tolerogenic dendritic cell; SEM, standard error of the mean. *P<0.001 compared to vehicle-treated mice.

• 

  Fig. 7 TolDC treatment recruits Tregs to the lung and the spleen. The lung and spleen cell preparations obtained from mice of each experimental group were stained with anti-CD4-APC and anti-CD25-FITC, and after permeabilization the cells were stained with anti-Foxp3-PE. (A) Gating on CD4+ T cells, and representative dot plots of lung Tregs (CD4+CD25highFoxp3+) and isotype controls. (B) Quantification of lung Tregs (CD4+CD25highFoxp3+) relative to total CD4+ T cells. (C) CD4+CD25highFoxp3+ splenocytes. (D) Confocal microscopy images showing CD3 (green), Foxp3 (red), and nuclei stained with DAPI (blue) in the lung tissue of TolDC-treated mice. Values are expressed as means±SEM of 5–7 mice per group. TolDC, tolerogenic dendritic cell; Treg, regulatory T cell; FITC, fluorescein isothiocyanate; DAPI, 4′,6-Diamidino-2-phenylindole; SEM, standard error of the mean. *P<0.05; †P<0.01; ‡P<0.001 (Bonferroni's multiple comparison test).

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