Indoleamine 2,3-Dioxygenase Is Not a Pivotal Regulator Responsible for Suppressing Allergic Airway Inflammation through Adipose-Derived Stem Cells

<div><p>Background</p><p>Although indoleamine 2,3-dioxygenase (IDO)-mediated immune suppression of mesenchymal stem cells (MSCs) has been revealed in septic and tumor microenvironments, the role of IDO in suppressing allergic airway inflammation by MSCs is not well documented. We evaluated the effects of adipose-derived stem cells (ASCs) on allergic inflammation in IDO-knockout (KO) asthmatic mice or asthmatic mice treated with ASCs derived from IDO-KO mice.</p><p>Methods and Findings</p><p>ASCs were injected intravenously in wild-type (WT) and IDO-KO asthmatic mice. Furthermore, asthmatic mice were injected with ASCs derived from IDO-KO mice. We investigated the immunomodulatory effects of ASCs between WT and IDO-KO mice or IDO-KO ASCs in asthmatic mice. In asthmatic mice, ASCs significantly reduced airway hyperresponsiveness, the number of total inflammatory cells and eosinophils in bronchoalveolar lavage fluid (BALF), eosinophilic inflammation, goblet hyperplasia, and serum concentrations of total and allergen-specific IgE and IgG1. ASCs significantly inhibited Th2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13, and enhanced Th1 cytokine (interferon-γ) and regulatory cytokines (IL-10, TGF-β) in BALF and lung draining lymph nodes (LLNs). ASCs led to significant increases in regulatory T-cells (Tregs) and IL-10⁺ T cell populations in LLNs. However, the immunosuppressive effects of ASCs did not significantly differ between WT and IDO-KO mice. Moreover, ASCs derived from IDO-KO mice showed immunosuppressive effects in allergic airway inflammation.</p><p>Conclusions</p><p>IDO did not play a pivotal role in the suppression of allergic airway inflammation through ASCs, suggesting that it is not the major regulator responsible for suppressing allergic airway inflammation.</p></div

The experimental protocol.

<p>(A) Mice were sensitized on days 0, 1, 7, and 8 by intraperitoneal injection of ovalbumin (OVA) and challenged intranasally on days 14, 15, 21, and 22. Purified adipose-derived stem cells (ASCs; 1 × 10⁶) derived from wild-type (WT) or indoleamine 2, 3-dioxygenase (IDO)-knockout (KO) mice were injected via the tail vein on days 12, 13, 19, and 20. (B) Mice were divided into three or four treatment groups.</p

Effect of adipose-derived stem cells (ASCs) on airway hyperresponsiveness (AHR) and inflammatory cells in the bronchoalveolar lavage fluid (BALF).

<p>ASCs significantly decreased AHR and the number of total inflammatory cells and eosinophils in asthmatic mice. Treatment with the PGE2 inhibitor or TGF-β neutralizing Ab eliminated the reduction in AHR (A, C) and total cell and eosinophil counts (B, D) induced by ASC treatment. Data are expressed as the mean ± SEM of four independent experiments each performed in triplicate. *,†,§,¶,††,ǁǁ,***,††† <i>p</i><0.001, ‡ <i>p =</i> 0.032, ǁ <i>p</i> = 0.045, ** <i>p</i> = 0.027, ‡‡ <i>p</i> = 0.032, §§ <i>p</i> = 0.005, ¶¶ <i>p</i> = 0.005.</p

The experimental protocol.

<p>(A) Mice were sensitized on days 0, 1, 7, and 8 by intraperitoneal injection of ovalbumin (OVA) and challenged intranasally on days 14, 15, 21, and 22. Purified adipose-derived stem cells (ASCs; 1 × 10⁶) were injected via the tail vein on days 12, 13, 19, and 20. PGE2 and TGF-β were blocked by intraperitoneal injection of a PGE2 inhibitor or anti-TGF-β-Ab on days 13, 14, 15, 16, 17, 20, 21, 22, and 23. (B) The mice were divided into five treatment groups.</p

Effect of adipose-derived stem cells (ASCs) on cytokine levels in the bronchoalveolar lavage fluid.

<p>IL-4, IL-5, and IL-13 were significantly higher in the OVA group than PBS group. ASC treatment significantly decreased IL-4, IL-5, and IL-13 but increased IL-10 and TGF—β in asthmatic mice. However, the PGE2 inhibitor (A) or TGF-β neutralizing Ab (B) eliminated these immunomodulatory effects of ASCs. Data are expressed as the mean ± SEM of four independent experiments each performed in triplicate. *,§,ǁ,¶,_**, §§§,§§§§, ¶¶¶¶¶ <i>p</i><0.001, †,_******<i>p</i> = 0.007, ‡ <i>p</i> = 0.027, ††,¶¶¶ <i>p</i> = 0.028, ‡‡ <i>p =</i> 0.030, §§ <i>p =</i> 0.010, ǁǁ <i>p</i> = 0.022, ¶¶ <i>p</i> = 0.032, ***,‡‡‡ <i>p</i> = 0.038, ††† <i>p =</i> 0.049, ǁǁǁ,†††† <i>p</i> = 0.003, ****,‡‡‡‡ <i>p =</i> 0.004, ǁǁǁǁǁ <i>p =</i> 0.026, ¶¶¶¶ <i>p</i> = 0.029, ***** <i>p</i> = 0.006, ††††† <i>p</i> = 0.012, ‡‡‡‡‡ <i>p</i> = 0.036, §§§§§ <i>p =</i> 0.042, ǁǁǁǁǁ <i>p =</i> 0.046.</p

Effects of adipose-derived stem cells (ASCs) on T-cells in the lung draining lymph nodes.

<p>The CD4⁺ T-cells were initially gated and the percentage of IL-4⁺, IFN-γ⁺, IL-10⁺, and CD25⁺ Foxp3⁺ T-cells subsequently analyzed. When treating asthmatic mice with ASCs derived from indoleamine 2, 3-dioxygenase (IDO)-knockout (KO) mice, the IL-4⁺ T-cell population decreased, but the IFN-γ⁺, IL-10⁺, and Foxp3⁺CD25⁺ T-cell populations increased.</p

Effects of adipose-derived stem cells (ASCs) on T cells in the lung draining lymph nodes.

<p>The CD4⁺ T cells were initially gated and the percentage of IFN-γ⁺, IL-4⁺, IL-10⁺, and CD25⁺ Foxp3⁺ T cells subsequently analyzed. When treating asthmatic mice with PGE2 inhibitor (A) or TGF-β neutralizing Abs (B), blocking of PGE2 and TGF-β prevented the increases in Foxp3⁺CD25⁺, IL-10⁺, and IFN-γ⁺ T cell populations and the decrease in the IL-4⁺ T cell population in the OVA+ASC group.</p

Effect of adipose-derived stem cells (ASCs) on cytokine levels in bronchoalveolar lavage fluid.

<p>IL-4, IL-5, and IL-13 levels were significantly higher in the OVA group than PBS group. ASCs (A) or ASCs derived from indoleamine 2, 3-dioxygenase (IDO)-knockout (KO) mice (B) treatment significantly decreased IL-4, IL-5, and IL-13, but increased IFN-γ, IL-10, and TGF-β in WT and IDO-KO asthmatic mice. Data are expressed as the mean ± SEM of four independent experiments performed in triplicate. _* <i>p</i><0.05, _** <i>p</i>≤0.005, _*** <i>p</i>≤0.001.</p

Effect of adipose-derived stem cells (ASCs) on serum levels of immunoglobulin.

<p>Systemic administration of ASCs resulted in a significant decrease in total and OVA-specific IgE in asthmatic mice. (A) A PGE2 inhibitor significantly increased OVA-specific IgE and IgG1 in the OVA+ASC group. (B) TGF-β neutralizing Ab resulted in significant increases in total IgE and IgG1 and OVA-specific IgE and IgG1 in the OVA+ASC group. Data are expressed as the mean ± SEM of four independent experiments each performed in triplicate. *,‡,ǁ,††,ǁǁ,†††,ǁǁǁ,†††† <i>p</i><0.001, †,*** <i>p =</i> 0.028, § <i>p =</i> 0.038, ¶,‡‡,¶¶ <i>p</i> = 0.008, **,§§ <i>p</i> = 0.009, ‡‡‡ <i>p</i> = 0.027, §§§ <i>p</i> = 0.037, ¶ <i>p</i> = 0.007, **** <i>p =</i> 0.035, ‡‡‡‡ <i>p</i> = 0.023, §§§§ <i>p</i> = 0.032.</p

Effect of adipose-derived stem cells (ASCs) on cytokine levels in the lung draining lymph nodes.

<p>ASCs (A) or ASCs derived from indoleamine 2, 3-dioxygenase (IDO)-knockout (KO) mice (B) treatment significantly decreased IL-4, IL-5, and IL-13 levels, but increased IFN-γ, IL-10, and TGF-β levels in WT and IDO-KO asthmatic mice. Data are expressed as the mean ± SEM of four independent experiments performed in triplicate. _* <i>p</i><0.05, _** <i>p</i>≤0.005, _*** <i>p</i>≤0.001.</p