Parasitic Nematode-Induced CD4⁺Foxp3⁺T Cells Can Ameliorate Allergic Airway Inflammation

<div><p>Background</p><p>The recruitment of CD4⁺CD25⁺Foxp3⁺T (T_reg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.</p><p>Methodology/Principal Findings</p><p>We compared the effects of T_reg cells from <i>Trichinella spiralis</i>-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced T_reg cells. After four weeks of <i>T. spiralis</i> infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4⁺Foxp3⁺ cells from <i>T. spiralis</i>-infected [Inf(+)Foxp3⁺] or uninfected [Inf(-)Foxp3⁺] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3⁺ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3⁺ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3⁺ cells migrated to inflammation sites in the lung and expressed higher levels of T_reg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3⁺ cells.</p><p>Conclusion/Significance</p><p><i>T. spiralis</i> infection promotes the proliferation and functional activation of T_reg cells. Parasite-induced T_reg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced T_reg cells. The adoptive transfer of Inf(+)Foxp3⁺ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.</p></div

Characterization of parasite-induced CD4⁺Foxp3⁺T cells.

<p>Splenocytes from <i>T. spiralis-infected</i> Foxp3 e GFP mice and normal Foxp3 e GFP mice. The GFP⁺ population was sorted using a FACSAria. After staining, the plots indicate the expression levels of several surface markers (related to T_reg function and activation) in gated CD4+ cell. The graphs showed mean value of the experimental group (A). Total RNA was extracted from Foxp3⁺ cells and cDNA was synthesized. The various gene expression levels in the lungs of each group were analyzed using real-time PCR. The GAPDH gene was used as a control. The expression of the important homing receptors, which are activation markers of T_reg cells, were analyzed (B). Purified splenocytes were cultured in 96-well round-bottomed plates in the presence of 1 µg/mL anti-CD3. Normal splenocytes were mixed in a 8∶1 ratio with CD4⁺Foxp3⁺T cells of parasite-infected and normal mice, in 200 µL of complete medium. After three days, cell viability was determined by trypan blue dye exclusion assay (C). (*<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001, <i>n</i> = 5 mice/group, three independent experiments).</p

Cytokine concentrations in BALF and OVA specific lymphocytes isolated from lung draining lymph node.

<p>Cytokine concentrations were measured in the BALF samples (A). The lymphocytes were activated by OVA. The wells was incubated with 1 µg/mL of OVA for 16 h at 4°C, and then the lymphocytes isolated from lung draining lymph node (LLN) were added to the well and incubated for three days. After activation, cytokine concentrations in the supernatant were measured using ELISA kits, in accordance with the manufacturer's instructions (B). [OVA-; PBS treated mice, OVA+; allergic airway inflammation-induced mice, IV(inf)+(-); CD4⁺Foxp3⁺T cell of normal mice adoptive transferred mice, IV(inf)+(+); CD4⁺Foxp3⁺T cell of <i>T. spiralis-infected</i> mice adoptive transferred mice, *<i>p</i><0.05, **<i>p</i><0.01, <i>n</i> = 6 mice/group, 3 independent experiments].</p

Recruitment of T_reg cells into spleen, lung draining lymph node, and lung with CD4⁺Foxp3⁺T cell adoptive transfer during asthma induction in mice.

<p>The lymphocytes were isolated from spleen and lung draining lymph node (A). After staining, the lymphocytes were firstly gated with CD4 and the percentage of CD25+Foxp3+ cells calculated using FACS analysis. Paraffin sections of the lung from all of the mice in each group receiving CD4⁺Foxp3⁺T cells (green) were immunofluorescently stained for CTLA-4 (red) and nuclei (DAPI, blue), representative pictures are shown, (white bar = 100 µm) (B). Population of CTLA4⁺Foxp3⁺ cells of each group were analyzed in the total lung cells based on analysis the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003410#pntd.0003410.s004" target="_blank">S4 Fig</a>. using Image J program(C). (The value in grape represented the number of the CTLA4⁺ Foxp3⁺ cells per 1×10⁵ DAPI positive cells) [a; OVA-IV(inf):+(-) group, b; OVA-IV(inf):+(+) group, c; OVA+IV(inf):+(-) group, d; OVA+IV(inf):+(+) group. Merge; merge of CTLA-4, GFP, and DAPI filed screens, Mag-Merge; magnification of Merge, Sp; spleen, LLN, lung draining lymph node, OVA-; PBS treated mice, OVA+; allergic airway inflammation-induced mice, IV(inf)+(-); CD4⁺Foxp3⁺T cell of normal mice adoptive transferred mice, IV(inf)+(+); CD4⁺Foxp3⁺T cell of <i>T. spiralis-infected</i> mice adoptive transferred mice, *<i>p</i><0.05, **<i>p</i><0.01, <i>n</i> = 6 mice/group, 3 independent experiments].</p

Recruitment of T_reg cells into spleen, lung draining lymph node, and the lung of the CD4⁺Foxp3⁺T cell adoptive transferred mice before asthma induction.

<p>The lymphocytes were isolated from spleen (A) and lung draining lymph node (B). Paraffin sections of lungs from mice receiving CD4⁺Foxp3⁺T cells (green) were immunofluorescently stained for CTLA-4 (red), and nuclei (DAPI, blue) representative pictures are shown, (white bar = 100 µm) (C). [a; OVA-IV(inf):+(-) group, b; OVA-IV(inf):+(+) group, c; OVA+IV(inf):+(-) group, d; OVA+IV(inf):+(+) group.] Population of CTLA4⁺Foxp3⁺ cells of each group were analyzed in the total lung cells based on analysis the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003410#pntd.0003410.s001" target="_blank">S1B Fig</a>. using Image J program (The value in grape represented the number of the CTLA4⁺ Foxp3⁺ cells per 1×10⁵ DAPI positive cells) (D). [Sp; spleen, LLN; lung draining lymph node, Merge; merge of CTLA-4, GFP, and DAPI filed screens, Mag-Merge; magnification of Merge, OVA-; PBS treated mice, OVA+; allergic airway inflammation-induced mice, IV(inf)+(-); CD4⁺Foxp3⁺T cell of normal mice adoptive transferred mice, IV(inf)+(+); CD4⁺Foxp3⁺T cell of <i>T. spiralis-infected</i> mice adoptive transferred mice, *<i>p</i><0.05, **<i>p</i><0.01, <i>n</i> = 6 mice/group, 3 independent experiments].</p

Experimental protocol of T_reg cell adoptive transfer and the airway inflammation induction model.

<p>The mice were divided into four experimental groups based on allergy induction and the adoptive transfer of T_reg cells isolated from <i>T. spiralis-infected</i> mice or uninfected mice. After four weeks of <i>T. spiralis</i> infection, T_reg (CD4⁺Foxp3⁺T) cells were isolated from spleen using a FACS sorter (A). Airway inflammation was induced via OVA-Alum or PBS sensitization and OVA or PBS challenge according to the experimental protocols given in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003410#s2" target="_blank">Methods</a> (B). (OVA-; PBS treated group, OVA+; allergic airway inflammation-induced group, OVA+IV(Inf)+(-); allergic airway inflammation-induced and CD4⁺Foxp3⁺T cell of normal mice adoptive transferred group, OVA+IV(Inf)+(+); allergic airway inflammation-induced and CD4⁺Foxp3⁺T cell of <i>T. spiralis-infected</i> mice adoptive transferred group. AI; after infection, IP; intra peritoneal, IN, intra nasal, IV; intra venous).</p