STAT6 regulates natural helper cell proliferation during lung inflammation initiated by Alternaria

Abstract

Asthma exacerbations can be caused by a number of factors, including the fungal allergen Alternaria, which is specifically associated with severe and near-fatal attacks. The mechanisms that trigger lung responses are unclear and might vary between allergens. A comparison between Alternaria, Aspergillus, Candida, and house dust mite, all allergens in humans, showed that only Alternaria promoted immediate innate airway eosinophilia within 12 h of inhalation in nonsensitized mice. Alternaria, but not the other allergens, induced a rapid increase in airway levels of IL-33, accompanied by IL-33 receptor (IL-33R)-positive natural helper cell (NHC) production of IL-5 and IL-13. NHCs in the lung and bone marrow constitutively expressed transcription factors [GATA-3 and E26 transformation-specific sequence-1 (ETS-1)] that could allow for rapid induction of T helper type 2 (Th2) cytokines. Lung NHC numbers and proliferation (%Ki-67), but not IL-5 or GATA-3 expression, were significantly reduced in STAT6-deficient mice 3 days after one challenge with Alternaria. Alternaria induced NHC expression of the EGF receptor ligand amphiregulin (partially dependent on STAT6), as well as EGF receptor signaling in the airway epithelium. Finally, human peripheral blood NHCs (CRTH2(+)CD127(+) lineage-negative lymphocytes) from allergic individuals highly expressed GATA-3 and ETS-1, similar to lung NHCs in mice. In summary, Alternaria-induced lung NHC proliferation and expression of amphiregulin are regulated by STAT6. In addition, NHCs in mouse and humans are primed to express Th2 cytokines through constitutive expression of GATA-3 and ETS-1. Thus several transcription factor pathways (STAT6, GATA-3, and ETS-1) may contribute to NHC proliferation and Th2-type responses in Alternaria-induced asthma.

Fig. 1.

Alternaria induces innate airway eosinophilia dependent on IL-33 receptor (IL-33R). A–C: C57BL/6 wild-type (WT) mice were given a single challenge of 100 μg of Alternaria extract or PBS intranasally, and bronchoalveolar lavage (BAL) eosinophils (Eos) and the cytokines IL-33 and IL-25 were measured (n = 4–14 mice per group). *P < 0.05, ***P < 0.005 vs. PBS, by Mann-Whitney test. D: BAL chemokines CCL11 and CCL24 12 h after Alternaria or PBS challenge (n = 4–14 mice per group). *P < 0.05 vs. PBS, by Mann-Whitney test. E–I: BAL IL-4, IL-5, IL-9, IL-13, and IFN-γ levels. ND, not detectable (n = 4–14 mice per group). *P < 0.05, ***P < 0.005 vs. PBS, except IL-5 (comparison between time points) by Mann-Whitney test. J: BAL eosinophils in mice treated with anti-IL-33R (anti-T1/ST2) antibody or control IgG for 2 days followed by a single challenge with Alternaria and analyzed 12 h later (n = 6 mice per group). ***P < 0.005, by Mann-Whitney test.

Fig. 2.

Alternaria, but not Aspergillus, Candida, or Dermatophagoides pteronyssinus house dust mite (HDM), induces an innate T helper type 2 (Th2)-like response. A: WT mice were given a single intranasal challenge with 100 μg of Alternaria, Aspergillus, Candida, HDM extract, or PBS, and levels of BAL eosinophilia were measured 12 h later (n = 4–8 mice per group). B–D: BAL IL-33 levels were measured 3 h and IL-5 and IL-13 12 h after Alternaria challenge (n = 4–8 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005, by Mann-Whitney test. Alternaria-challenged mice were independently compared with Aspergillus, Candida, and HDM groups.

Fig. 3.

Alternaria stimulates lung natural helper cells (NHCs) in vivo to produce Th2 cytokines. C57BL/6 WT mice were given 1 (A–C) or 4 (D and E) intranasal challenges with Alternaria or PBS, and lungs were processed for fluorescein-activated cell sorting analysis. Single-cell suspensions from lung were stained for CD45, T1/ST2, and lineage markers CD3, Gr-1, CD11b, B220, Ter-119, CD11c, NK1.1, CD4, CD5, CD8, FcεR1, TCRβ, TCRγδ, and CD19. A: cells were gated on CD45-positive cells, lymphocytes, and lineage-negative T1/ST2-positive cells; cytospin of sorted lineage-negative T1/ST2-positive cells is shown at bottom left (scale bar, 10 μm). FSC and SSC, forward and side scatter. B: surface expression of markers Sca-1, c-Kit, IL-7 receptor (IL-7R), Thy1.2, CD25, and CD44. Solid gray peak represents isotype control staining. C: lineage-negative CD45-positive T1/ST2-positive lymphocytes from lungs of mice exposed to PBS or Alternaria were gated and analyzed for IL-5 and IL-13 production or isotype staining. D: total lung NHCs after 1 and 4 challenges with PBS or Alternaria (left) and NHC intracellular IL-5 after 4 challenges with PBS or Alternaria (right). Results in A–D are representative of 2–3 independent experiments and pooled lungs from 2–4 mice per group. E: BAL eosinophils and serum IgE after 4 challenges with Alternaria or PBS (n = 4 mice per group). *P < 0.05, **P < 0.01, by Mann-Whitney test.

Fig. 4.

NHCs express GATA-3 and ETS-1 constitutively independent of STAT6. A and B: single-cell suspensions from lung and bone marrow of naive mice were stained for surface lineage, CD45, IL-33R (T1/ST2), and intracellular ETS-1 or GATA-3. Lineage-positive and -negative T1/ST2-positive lymphocytes (left) were gated, and ETS-1 and GATA-3 expression was determined (middle and right). C: intracellular staining of single-cell suspensions from naive WT and STAT6^−/− mouse lungs for GATA-3 and ETS-1 after gating on lineage-negative T1/ST2-positive cells. Solid gray peaks represent isotype staining. D: WT and STAT6^−/− NHC intracellular IL-5 production from naive mice and 12 h after Alternaria challenge (Alt). Results are representative of 2–3 independent experiments; lungs and bone marrow were pooled from 2–4 mice per group.

Fig. 5.

STAT6 regulates lung NHC proliferation and innate eosinophilia after Alternaria challenge. A: total lung NHCs in single-cell suspensions from WT and STAT6^−/− mice before challenge (naive), as well as 12 h and 3 days after Alternaria challenge. B: lung NHCs from naive mice and 3 days after Alternaria challenge (Alt) were stained for surface IL-4Rα and IL-13Rα₁. Results from 1 of 2 representative independent experiments are shown for each time point (A and B). C: lung NHCs from WT and STAT6^−/− naive mice and 3 days after Alternaria challenge were stained for Ki-67. Results are from 3 independent experiments; lungs were pooled from 2 WT and STAT6^−/− mice. D: total BAL eosinophils from naive WT and STAT6^−/− mice as well as 3 days after Alternaria challenge (n = 7–8 mice per group). **P < 0.01, by Mann-Whitney test. E: BAL CCL11 and CCL24 at 12 h (n = 7–11 mice per Alternaria group and 3 mice per PBS group). *P < 0.05, **P < 0.01, by Mann-Whitney test.

Fig. 6.

IL-7R is required for lung NHCs and Alternaria-induced innate eosinophilia. WT, RAG2^−/−, and IL-7R^−/− mice were challenged with 100 μg of Alternaria or PBS and analyzed 3 or 12 h later. A: presence of lung NHCs in unchallenged mice. B: BAL eosinophils and IL-5 levels 12 h after Alternaria or PBS challenge in WT or IL-7R^−/− mice. C: BAL eosinophils and IL-5 levels 12 h after Alternaria or PBS challenge in RAG2^−/− and IL-7R^−/− mice (n = 4 mice per group). *P < 0.05, by Mann-Whitney test. D: percentage of lung eosinophils at 3 h after challenge, gated on CD45⁺ cells. Results are from 3 lungs pooled in Alt groups and 1 lung in PBS group.

Fig. 7.

NHC amphiregulin expression and EGF receptor (EGFR) signaling are induced after Alternaria challenge. A: BAL amphiregulin (AREG) in WT mice challenged with PBS, Alternaria, Aspergillus, Candida, or HDM (top) and ELISA of BAL amphiregulin levels after Alternaria challenge (bottom) (n = 4–8 mice per group). *P < 0.05 vs. PBS, ***P < 0.0005, by Mann-Whitney test. B: immunostaining for EGFR and phosphorylated EGFR (phospho-EGFR) 12 h after PBS or Alternaria challenge (scale bars, 50 μm) and number of positive cells per airway (n = 13–19 airways per group). ****P < 0.0001 vs. PBS, by Mann-Whitney test. C and D: NHC surface amphiregulin expression 6 h after challenge with Alternaria or PBS and 12 h after challenge with PBS, Alternaria, Aspergillus, Candida, or HDM. E: NHC surface amphiregulin in WT and STAT6^−/− mice 12 h after Alternaria challenge. Surface staining from pooled lungs is representative of 2 independent experiments. Solid gray peaks represent isotype staining.

Fig. 8.

Human lineage-negative CRTH2⁺CD127⁺ lymphocytes from allergic individuals express GATA-3 and ETS-1. A: human peripheral blood mononuclear cells from allergic patients were stained for lineage, lineage-negative population was gated and analyzed for CRTH2 and CD127 expression or isotype, and lineage-negative CRTH2⁺CD127⁺ cells were analyzed for intracellular GATA-3 and ETS-1 expression. Results are representative of 6 patients. B: human peripheral blood mononuclear cells stained for lineage (Lin), CD127, CRTH2, and GATA-3. Percentage of GATA-3-positive lineage-positive and lineage-negative CD127⁺ cells and GATA-3-positive lineage-positive and lineage-negative CRTH2⁺ cells (n = 6 allergic individuals). *P < 0.05, **P < 0.005, by Mann-Whitney test.