Transfer of in vivo primed transgenic T cells supports allergic lung inflammation and FIZZ1 and Ym1 production in an IL-4Rα and STAT6 dependent manner

Abstract

Background: CD4+ T helper type 2 (TH2) cells, their cytokines IL-4, IL-5 and IL-13 and the transcription factor STAT6 are known to regulate various features of asthma including lung inflammation, mucus production and airway hyperreactivity and also drive alternative activation of macrophages (AAM). However, the precise roles played by the IL-4/IL-13 receptors and STAT6 in inducing AAM protein expression and modulating specific features of airway inflammation are still unclear. Since TH2 differentiation and activation plays a pivotal role in this disease, we explored the possibility of developing an asthma model in mice using T cells that were differentiated in vivo.

Results: In this study, we monitored the activation and proliferation status of adoptively transferred allergen-specific naïve or in vivo primed CD4+ T cells. We found that both the naïve and in vivo primed T cells expressed similar levels of CD44 and IL-4. However, in vivo primed T cells underwent reduced proliferation in a lymphopenic environment when compared to naïve T cells. We then used these in vivo generated effector T cells in an asthma model. Although there was reduced inflammation in mice lacking IL-4Rα or STAT6, significant amounts of eosinophils were still present in the BAL and lung tissue. Moreover, specific AAM proteins YM1 and FIZZ1 were expressed by epithelial cells, while macrophages expressed only YM1 in RAG2-/- mice. We further show that FIZZ1 and YM1 protein expression in the lung was completely dependent on signaling through the IL-4Rα and STAT6. Consistent with the enhanced inflammation and AAM protein expression, there was a significant increase in collagen deposition and smooth muscle thickening in RAG2-/- mice compared to mice deficient in IL-4Rα or STAT6.

Conclusions: These results establish that transfer of in vivo primed CD4+ T cells can induce allergic lung inflammation. Furthermore, while IL-4/IL-13 signaling through IL-4Rα and STAT6 is essential for AAM protein expression, lung inflammation and eosinophilia are only partially dependent on this pathway. Further studies are required to identify other proteins and signaling pathways involved in airway inflammation.

Figure 1

Cytokine production by unimmunized and OVA/Alum immunized D011.10xRAG2^-/- mice. Splenocytes isolated from either naïve (unimmunized) or OVA/Alum immunized D011.10xRAG2^-/- mice were cultured in media containing 20 U/ml IL-2 and were either stimulated with PMA/Ionomycin (PMA/Ion) or left untreated for 18 h. IL-4 and IL-5 secretion into the cell culture supernatant was quantitated using ELISA. Data represented as concentration of cytokine ± SEM in ng/ml. n = 3 for each group. * p < 0.05.

Figure 2

Comparison of proliferation and activation status of naïve vs. in vivo primed T cells. (A) Schematic representation of the protocol used in this experiment. Briefly, 1.5 × 10⁶ naïve or in vivo primed CD4+ T cells were adoptively transferred into STAT6xRAG2^-/- mice and primed with OVA/alum i.p. on day 1. One group of mice was treated daily with BrdU (1 mg/mouse) i.p for 3 days before harvesting spleens on day 5. Splenocytes were pooled together and total cell counts were recorded. Cells were stained with anitbodies to CD4, KJ126, CD44 and BrdU and flow cytometry was performed. Another group of mice, that didn't receive BrdU were immunized with OVA/alum a second time on day 8. Four days later, splenocytes were harvested, counted and stimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 6 h. (B) BrdU and CD44 expression in the CD4+ KJ126+ population in the naïve T cell or in vivo primed T cell transfer groups are shown. (C) CD44 expression in the CD4+ KJ126+ population in naïve vs. in vivo primed T cell transfer groups on day 12 is shown. IL-4 production by naïve and in vivo primed DO11.10 CD4 T cells was measured by intracellular cytokine staining (ICS).

Figure 3

Degree of BAL eosinopilia, cytokine and chemokine secretion in RAG2^-/- STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice. The asthma protocol used in this study is depicted in (A). In vivo primed DO11.10+ CD4+ T cells were adoptively transferred into RAG2^-/-, STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice. Mice were primed with either alum or 100 μg of Ova in alum i.p on d. 1 & 6 and then challenged with nebulized PBS or 1% Ova in PBS on d. 12 &14. BAL fluid was recovered 48 h after the last challenge and cells were analyzed by differential staining. Lung tissue was collected for histological analysis. (B) The total number of cells (T), macrophages (M), eosinophils (E), lymphocytes (L) and polymorphoneutrophils (P) present in the BAL in RAG2^-/-, STAT6xRAG2^-/- and IL-4RαxRAG2^-/- mice are represented here in the form of bar graphs. Data represented as numbers ± SEM. * (p < 0.05), represents statistically significant differences between the OVA and Alum treated mice in each group. n = 5 for Ova treated mice, n = 3 for alum treated. (C) Chemokine and cytokine levels in BAL samples from OVA primed and challenged RAG2^-/-, STAT6xRAG2^-/- and IL-4RαxRAG2^-/- mice were analyzed using a multiplex array system. Data are presented as mean chemokine or cytokine level in pg/ml ± SEM. * p < 0.05; ** p < 0.01; + p < 0.0001. n = 4 for RAG2^-/- mice, n = 3 each for STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice. Representative data from one of three independent experiments is shown.

Figure 4

CD3+ T cells migrate into the lung in absence of STAT6. Allergic lung disease was induced in RAG2^-/-, STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice as described above. Images (10×, 40× and 100× magnifications) of representative lung sections stained with antibodies to CD3 are shown in (A). CD3+ cells appear brown. Panels a-c: RAG2^-/- lung sections; d-f: STAT6xRAG2^-/- sections and g-i: IL-4RαxRAG2^-/- sections. n = 5 for each mouse strain. (B) Graphical representation of the immunohistochemistry data shown above. Number of CD3+ cells in each lung section was counted and graphed. Data represented as cell counts ± SEM. HPF: high power field; 100×. * p < 0.05.

Figure 5

FIZZ1 and YM1 expression in the lung is dependent on STAT6 and IL-4Rα. Allergic lung inflammation was induced in RAG2^-/-, STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice as mentioned in Figure 3 and materials and methods. FIZZ1 and YM1 expression was analyzed in serial sections of mouse lungs by immunohistochemistry. Photomicrographs of FIZZ1 and YM1 expression in epithelial cells (A) and macrophages (B) in representative lung sections are shown. (C) YM1 expression in multinucleate giant cells (MNG) in RAG2^-/- mice. Images in (B) and (C) are of 100× magnification.

Figure 6

Presence of FIZZ1 and YM1 protein in BAL fluid. BAL fluid samples from RAG2^-/-, STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice treated as described in Figure 4 were collected. FIZZ1 and YM1 protein secreted into the BAL fluid in the three groups of mice was detected by western blotting (A). Equal amounts of total protein were loaded into every well. Each lane represents an individual mouse. Densitometry analysis was performed on the autoradiograms from each blot and the values are represented on a graph (B). White bars represent densitometry values for FIZZ1, black bars represent YM1. * p < 0.01; # p < 0.001. n = 3 for each group.

Figure 7

Reduced airway remodeling in mice deficient in STAT6 and IL-4Rα. RAG2^-/-, STAT6xRAG2^-/- or IL-4RαxRAG2^-/- mice were subjected to the asthma protocol described in Figure 3. (A) Paraffin embedded lung sections from each group of mice were stained with Masson's Trichrome. Keratin and muscle fibers are stained in red, collagen in blue, cytoplasm in light red/pink and nuclei in black. Photomicrographs of collagen deposition around the airways (panels a-c) or blood vessels (panels d-f) were collected at 10× (panels a-f) and 100× (inset) magnification. Photomicrographs (100×) of the airway smooth muscle (ASM) layer in H&E stained lung sections from each mouse group is shown in panels g-i. Arrows depict the thickness of the ASM layer (transverse section). (B) The amount of collagen present in the lung was quantified using NIH Image J software. Data is represented as area of collagen (blue stain) ± SEM. # p < 0.001. n = 20 airways/blood vessels per group. (C) The distance between the innermost aspect and outermost aspect of the smooth muscle was measured at 3 different positions around each airway, using NIH Image J software. Data is represented as airway smooth muscle thickness in μm ± SEM. * p < 0.01. An average of 30 airways was used for each group.