STAT6 expression in multiple cell types mediates the cooperative development of allergic airway disease

Abstract

Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6(-/-) mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6(-/-) mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6(-/-) mice even when STAT6(+/+) BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2(-/-) mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4(+)CD25(+)Foxp3(+) T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6(-/-) mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6(-/-) mice were equally efficient in suppressing CD4(+) T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.

FIGURE 1

Experimental protocols used in this study (A) and a schematic representation of the BM chimeras (B). A and B, BM chimeras were generated by transferring BM isolated from WT, STAT6^−/−, Rag2^−/−, and STAT6×Rag2^−/− mice into sublethally irradiated recipient mice as noted. Six weeks later, the mice received a priming injection of OVA in alum. In some cases, the mice received OVA-specific Th2 effectors derived from DO11.10 mice (as described in Materials and Methods) prior to immunization. A booster injection of Ag was administered 2 wk later. Then, the mice were challenged with aerosolized Ag on days 19, 21, and 26. Twenty-four hours after the last challenge, AHR measurements were performed. BAL was performed on euthanized mice 24 h later. Of note, nonchimeric groups of mice were treated with OVA as described above with omission of the irradiation and BM transfer steps.

FIGURE 2

Alternative lung pathology in allergen-treated STAT6^−/− mice (A, B). A, WT, Rag2^−/−, STAT6^−/−, and STAT6×Rag2^−/− mice were immunized with OVA as described in Fig. 1A. BAL fluid was harvested, and the cells were analyzed by differential counting after cytospin. The average numbers (n = 3 to 5) of total cells (white bars), macrophages (black bars), eosinophils (right hatched bars), lymphocytes (left hatched bars), and neutrophils (gray bars) ±SEM are shown. *, ¶, **, †: p < 0.02 (differences in absolute numbers of total cells, eosinophils, lymphocytes, and neutrophils, respectively, in BAL fluid between OVA-treated WT and STAT6^−/− mice). B, Lung sections were stained with H&E or PAS (inset) (original magnification ×4; insets ×100). *The percentage of eosinophils in the BAL fluid was calculated.

FIGURE 3

STAT6 regulates inflammatory macrophage accumulation in the lung tissue. WT, Rag2^−/−, STAT6^−/−, and STAT6×Rag2^−/− mice were immunized with OVA as described in Fig. 1A. Control animals were injected with alum alone and challenged with PBS. A–C, Lung sections were stained with anti-F4/80, anti–Mac-2, or anti–YM-1 as noted and analyzed by immunohistochemistry (original magnification ×20; insets for Mac-2 are ×40). D and E, WT, STAT6^−/−, and STAT6×Rag2^−/− mice were immunized with OVA as described in Fig. 1A. Isolated lungs were subjected to enzymatic digestion to obtain single-cell suspensions. The cells were then stained with anti-CD11b and anti-F4/80 plus anti–MHC-II (D) or with anti-CD11b and anti-CD11c plus anti–MHC-II (E).

FIGURE 4

STAT6 deficiency affects the baseline lung responsiveness to methacholine. WT and STAT6^−/− mice were immunized with OVA as described in Fig. 1A. Control mice were left untreated. Mice were exposed to increasing concentrations of methacholine, and the change in PenH was measured using whole-body plethysmography. The percentage change in PenH is shown (meant ± SEM). *p < 0.05 (OVA-treated WT versus STAT6^−/− mice).

FIGURE 5

STAT6-dependent allergic airway inflammation. The indicated BM chimeras were generated as described in Materials and Methods and depicted in Fig. 1B. Where indicated, OVA-specific Th2 effectors were transferred by i.v. injection 1 d prior to immunization with OVA/alum. A, Lungs were lavaged, and the percentage of eosinophils in the BAL (*) was determined by differential counting after cytospin. Lungs were harvested and stained with H&E (original magnification ×20; insets ×100). B, Cytokines and chemokines in the BAL fluid were measured using the Pierce Searchlight array. Data shown are mean ± SEM (n = 3 to 4 mice/group). *, **, ¶: p < 0.05 [WT(d)-WT(r) mice versus WT(d)-STAT6^−/−(r), Rag2^−/−(d)-STAT6^−/−(r), and WT(d)-Rag2× STAT6^−/−(r) mice, respectively].

FIGURE 6

STAT6-independent allergic airway inflammation (A, B). The indicated BM chimeras were generated as described in Materials and Methods and depicted in Fig. 1B. Non-irradiated WT mice and the chimeras were immunized with alum or OVA/alum and challenged as indicated. A, Lungs were lavaged, and the cells in the BAL fluid were analyzed by differential counting. The average numbers of eosinophils are shown ± SEM (n = 3 to 4 mice/group). B, Representative lung histology (H&E, original magnification ×20; left insets ×100) from OVA-primed mice is shown. Right insets, PAS, original magnification ×40. *Percentage of eosinophils in BAL fluid. C, BAL cytokines and chemokines were measured as described in Materials and Methods. Data shown are mean ± SEM (n = 3 to 4 mice/group). *p < 0.05 [levels of BAL IL-4, TGF-β1, and eotaxin in Rag2×STAT6^−/−(d)-Rag2×STAT6^−/−(r) mice versus all other experimental groups of mice with an exception for comparable BAL TGF-β1 in Rag2^−/−(d)-Rag2×STAT6^−/−(r) mice]; **p < 0.05 [Rag2^−/−(d)-Rag2^−/−(r) mice versus WT mice]; ^†p < 0.05 [Rag2^−/−(d)-Rag2×STAT6^−/−(r) versus WT mice]; ^¶p < 0.05 [Rag2^−/−(d)-Rag2×STAT6^−/− (r) mice versus WT mice].

FIGURE 7

STAT6 deficiency alters the number of Tregs in the lung. WT and STAT6^−/− mice were immunized with alum or OVA/alum as described in Fig. 1A. The mice were challenged as indicated. Lungs were digested with collagenase–DNAse, and cell suspensions were analyzed by FACS for expression of CD4 and CD8 on T cells (A) and for expression of CD4, CD25, and Foxp3 (B). Foxp3 staining in the CD4⁺, CD25^hi population is shown.

FIGURE 8

STAT6-deficient Tregs are functionally active. Tregs were obtained from the spleens of untreated WT (open bars) and STAT6^−/− (black bars) mice as described in Materials and Methods. CD4⁺CD25⁻ WT T cells were obtained and used as effectors. The effector T cells were incubated with increasing amounts of WT or STAT6^−/− Tregs. Cells were stimulated with Dynabeads (CD3/CD28) for 72 h, and ³H was added to the cultures for the last 24 h before cell harvest. Data are presented as mean cpm ± SEM of ³H incorporation in 72-h cultures.