Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell–mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca2+ and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11chighCD11blow than CD11clowCD11bhigh DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non–Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non–Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO–induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.

We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells and CD4+CD25+ICOS+Foxp3+IL-10+ T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)–sensitized and –challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 μg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)–β in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-β levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4+IL-17+IL-23R+CD25− T cells with high expression of retinoic acid–related orphan receptor (ROR)–γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4+IL-17+IL-23R+CD25− T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-β levels in the BALF, which, in turn, decrease CD4+IL-17+IL-23R+ROR-γt+CD25− T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice.

We recently reported that the adoptive transfer of T-regulatory cells (Tregs) isolated from lung and spleen tissue of green fluorescent protein–transgenic mice reversed airway hyperresponsiveness and airway inflammation. Because Programmed Death-1 (PD-1) is a pivotal receptor regulating effector T-cell activation by Tregs, we evaluated whether PD-1 is involved in the therapeutic effect of naturally occurring Tregs (NTregs) and inducible Tregs (iTregs) in cockroach (CRA)-sensitized and challenged mice. The CD4+CD25+ NTregs and CD4+CD25− iTregs isolated from the lungs and spleens of BALB/c mice were adoptively transferred into CRA-sensitized and CRA-challenged mice with and without anti–PD-1 antibody (100 μg/mice). The CD4+CD25+ T cells in the lung were phenotyped after adoptive transfer. Concentrations of IL-4, IL-5, IL-10, IFN-γ, and IL-13 in bronchoalveolar lavage fluid (BALF) were measured using ELISA. The NTregs and iTregs from either lung or spleen tissue reversed airway hyperresponsiveness for at least 4 wk. However, the therapeutic effect was blocked by administering the anti–PD-1 antibody. The administration of Tregs-recipient mice with anti–PD-1 antibody significantly decreased cytotoxic T-lymphocyte antigen-4 expression, with low concentrations of Forkhead-winged transcriptional factor box 3 (Foxp3) mRNA transcripts in lung CD4+CD25+ T cells. These mice had substantially higher concentrations of BALF IL-4, IL-5, and IL-13, but significantly decreased levels of BALF IL-10. Adoptive therapy recipients without the anti–PD-1 antibody exhibited high levels of CTLA-4 expression and Foxp3 transcripts in lung CD4+CD25+ T cells, with a significant decrease in BALF IL-4, IL-5, and IL-13 concentrations and a substantial increase in BALF IL-10 concentrations. These data suggest that the reversal of airway hyperresponsiveness and airway inflammation by Tregs is mediated in part by PD-1, because other costimulatory molecules (e.g., inducible costimulatory molecule [ICOS] or CTLA-4) have been shown to play a role in Treg-mediated suppression.

Advances have been made in defining the mechanisms for the control of allergic airway inflammation in response to inhaled antigens. Several genes, including ADAM33, DPP10, PHF11, GPRA, TIM-1, PDE4D, OPN3, and ORMDL3 have been implicated in the pathogenesis and susceptibility to atopy and asthma. Growing evidence associates asthma with a systemic propensity for allergic type 2 T-cell cytokines. Disordered coagulation and fibrinolysis also exacerbate asthma symptoms. Balance among functionally distinct dendritic cell subsets contribute to the outcome of T cell-mediated immunity. Allergen-specific T-regulatory cells play a pivotal role of in the development of tolerance to allergens and immune suppression. Major emphasis on immunotherapy for asthma during the past decade has been to direct the immune response to a type 1 response or immune tolerance. In this review article, we discuss the current information on the pathogenesis of allergic airway inflammation and potential immunotherapy, which could be beneficial in the treatment of airway inflammation, allergy, and asthma.

Background

Dendritic cell subsets display different functional role in regulating immune response and lead to various outcomes including Th1 versus Th2 or regulatory versus immunologic response. Administration of Flt3-Ligand prevents and reverses allergic airway inflammation and airway hyperresponsiveness in a mouse model. However, the underlying mechanisms are unclear.

Objective

We characterized and examined the role of lung dendritic cell subsets in the therapeutic effect of Flt3-Ligand.

Methods

Dendritic cells were isolated from the lungs of OVA-sensitized and challenged mice treated with rhFlt3-Ligand. Two populations of CD11c+ cells labeled with fluorochrome-conjugated antibodies were sorted. The ability of the purified cells to stimulate T cell proliferation and cytokine secretion pattern by different DC subsets was examined. Also, dendritic cells were adoptively transferred in mice to examine their effect on pulmonary function.

Results

Two dendritic cell populations, CD11chighCD11blow and CD11clowCD11bhigh, were identified in the lungs of naïve and OVA-sensitized and challenged mice with and without treatment with Flt3-Ligand. The expression levels of CD8α, B220, CD19, F4/80, MHC II, CCR7, CD40, PDL1, PDL2, CD80, and CD86 were distinctly different between the two DC populations, which supports the notion that, CD11chighCD11blow and CD11clowCD11bhigh dendritic cells, potentially have regulatory and immunogenic properties, respectively. Administration of Flt3-Ligand increased the dendritic cells with regulatory potential in the lungs of antigen-sensitized mice, and CD11chighCD11blow dendritic cells acquired a maximum degree of regulatory capacity after Flt3-Ligand treatment.

Conclusion

These data suggest that Flt3-Ligand reverses airway hyperresponsiveness by regulating the function of lung dendritic cells in a mouse model of allergic airway inflammation.

Clinical implication

Flt3-Ligand could be a potential immunomodulator in the treatment of established asthma.