T-Regulatory Cells and FLT3-Ligand in the Therapy of Cockroach Antigen-induced Asthma

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McGee, Halvor Sim
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Asthma is a chronic disease of the airways, characterized by airway hyper-responsiveness (AHR) and airway inflammation. Many inflammatory cells, including mast cells, eosinophils, neutrophils, and lymphocytes, are critical in the initiation of the pathophysiological changes in asthma. The mobilization and recruitment of these inflammatory cells are primarily due to the release of proinflammatory cytokines (IL-4, IL-5, IL-9 and IL-13) released from TH2 cells. Normally, TH1 responses are presumed to protect from invasive bacterial, protozoa and viral infections, while TH2 responses protect from extracellular, parasites and helminths. One mechanism by which the ratio of TH1 and TH2 cells is maintained is by the immune T cell subset T-regulatory cells (Tregs) (CD4+CD25+). Tregs are divided into two categories: “naturally occurring” CD4+CD25+ T regulatory cells (NTregs), that constitutively express Foxp3 and CD25, and “inducible” CD4+CD25- T regulatory cells (iTregs), which up-regulate CD25 and Foxp3 after exposure to inhaled antigen in the lung. However, how these cells regulate allergic airway inflammation and AHR in asthma remains ill-defined.|In this study, the effect of NTregs and iTregs, isolated from mouse lung and spleen (BALB/c), was examined by adoptive transfer into cockroach (a common inner-city allergen)-sensitized and challenged mice. The adoptive transfer of either of the two subtypes of Tregs reversed AHR and airway inflammation. The cells from the spleen had longer-lasting effect than those of lung cells. Tregs isolated from the lungs of the adoptively transferred recipients expressed significantly high levels of IL-10, TGF-β, and Foxp3 transcripts. Bronchoalveolar lavage fluid (BALF) of adoptively-transferred mice had decreased levels of Th2 cytokines, IL-4, IL-5, IL-13, and increased levels of IL-10 compared to cockroach-sensitized and challenged mice without adoptive transfer. The lung T cells isolated from the adoptively-transferred recipients expressed significantly higher levels of programmed death-1 (PD-1), a negative regulator of immune responses. These data suggest a critical role of PD-1 in the reversal of AHR and airway inflammation by Tregs. To confirm the role of PD-1, the effect of anti-PD-1 antagonist was examined. The effects of the adoptive transfer of Tregs were blocked after administration of the anti-PD-1 antagonist. Tregs isolated from the lungs of the mice that received the anti-PD-1 antagonist exhibited a significant decrease of Foxp3 transcripts, an increase in BALF IL-4, IL- 5 and IL-13 levels, and a decrease in BALF IL-10 levels. Additionally, CTLA-4, a negative regulatory of T cell activation, was significantly decreased in the lung Tregs of mice that received the anti-PD-1 antagonist.|Flt3-ligand (Flt3-L), a hematopoietic growth factor, dramatically increased the number of dendritic cells in the lungs of Ova-sensitized and challenged mice. Therefore, the effect of Flt3-L on CD4+CD25+ T cells in cockroach-sensitized and challenged mice was examined. These mice received three i.p. injections of anti CD25 antibody (PC61) (250 μg) and Flt3-L (3μg) daily for ten days. Cytokines and immunoglobulins levels in the serum were measured and differential BALF cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-γ, eosinophilia and substantially increased IL-10 and the number of CD4+CD25+Foxp3+IL-10+ T-cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, BALF IL-5 and IFN-γ levels and decreased BALF IL-10 levels and the number of CD4+CD25+Foxp3+IL-10+ T-cells. Flt3-L significantly decreased the expression of cell surface receptor CD62L, increased expression of cell surface receptor ICOS and Foxp3 transcripts in CD4+CD25+ T-cells isolated from lungs of CRA-sensitized and challenged mice. Flt3-L significantly inhibited the effect of CRA sensitization to decrease GATA3 expression in lung CD4+CD25+ T-cells.|Finally, the functional effect and migration of Tregs was examined by adoptively transferring naturally occurring CD4+CD25+ T- regulatory cells (NTregs) and CD4+CD25- inducible T-regulatory cells (iTregs) from lung and spleens of GFP-transgenic BALB/c mice into cockroach-sensitized and challenged mice. Adoptive transfer of either Ntregs or iTregs from lung or spleen reversed airway inflammation and AHR to methacholine, and the effect lasted for at least four weeks. GFP-labeled iTregs up-regulated CD25 and Foxp3, and migrated to lymph node and lung. Lung CD4+CD25+ T-cells isolated from each group of recipients mice were ICOShigh and PD-1-positive; however, higher expression of PD-1 was found in the spleen iTregs (S25-) and lung iTregs (L25-) groups. Higher levels of TGF-β and IL-10 transcripts, and BALF IL-10 and INF-γ levels were observed in lung CD4+CD25+ cells from the L25- and S25- cell-recipient mice than from lung NTregs (L25+) and spleen NTregs (S25+) cell recipient mice. Adoptive transfer of either cell type significantly reduced BALF IL-4, IL-5 and IL-13 levels.|In conclusion, the findings from this study demonstrated the potential therapeutic properties of T-regulatory cells and the underlying cellular mechanisms in reversing the deleterious effects of allergen-induced airway inflammation and AHR in a murine model of cockroach antigen-induced asthma. Furthermore, the immunomodulatory effect of Flt3-L in allergic asthma involves an increase in lung CD4+CD25+ICOS+Foxp3+ T cells with a decrease in CD4+CD25+CD62L- T-cells. The findings from this study may be useful in developing better therapeutic modalities applicable to allergic asthma.
Creighton University
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