CD11b+ myeloid cells are the key mediators of Th2 cell homing into the airway in allergic inflammation

Abstract

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b(+) myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b(+) myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

Figure 1. STAT6 expression in airway lining cells does not support Th2 cell and eosinophil trafficking into the airways

A) Number of CD4⁺ and KJ⁺ OVA-specific Th2 cells, and B) CD4⁺ and CD8⁺ T cells in the BAL of wild-type, STAT6^-/- and EpiSTAT6 mice following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA or PBS challenges (n = 4-6 mice per group, from 2 experiments). C) Percentage and D) number of eosinophils in the BAL of wild-type, STAT6-/- and EpiSTAT6 mice following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 6 mice per group, from 2 experiments). E) Representative lung histology (stained with hematoxylin and eosin) from wild-type (i, iv - 100x and 400x respectively), STAT6^-/- mice (ii, v), and EpiSTAT6 (iii, vi) mice following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 6 mice per group, from 2 experiments). Black bars are 100 μm, arrows demonstrate clusters of eosinophils only in the section from wild-type lungs. F) Lung chemokine RNA copies normalized to copies of GAPDH RNA and G) BAL chemokine protein levels in wild-type, STAT6^-/- and EpiSTAT6 mice following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA or PBS challenges (n = 4-6 mice per group, from 2 experiments).

Figure 2. Mucus hyper-secretion in wild-type mice, STAT6^-/- mice, EpiSTAT6 mice, and STAT6^-/- mice following bone marrow transplant

Representative histologic sections stained with PAS to evaluate mucus hypersecretion following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges. Sections are from wild-type BALB/c mice (A), STAT6^-/- mice (B), EpiSTAT6 mice (C), wild-type BALB/c mice after bone marrow transplant with wild-type bone marrow (D), STAT6^-/- mice after bone marrow transplant with wild-type bone marrow (E), or STAT6^-/- mice after bone marrow transplant with RAG1^-/- bone marrow (F). Black bars are 100 μm.

Figure 3. Intratracheal transfer of Th2 cells does not restore eosinophil trafficking into the airways in STAT6^-/- mice

A) Representative flow cytometry of BAL cells stained for CD4 and KJ following intratracheal transfer of OVA-specific in vitro polarized Th2 lymphocytes and 3 OVA challenges (n = 6 mice per group, from 2 experiments). B) Number of CD4⁺ and KJ⁺ OVA-specific Th2 cells in the BAL of wild-type and STAT6^-/- mice following intratracheal transfer of OVA-specific in vitro polarized Th2 lymphocytes and 3 OVA challenges (n = 6 mice per group, from 2 experiments). C) Number of eosinophils in the BAL of wild-type and STAT6-/-mice following intratracheal transfer of OVA-specific in vitro polarized Th2 lymphocytes and 3 OVA challenges (n = 6 mice per group, from 2 experiments). D) Percentage of CCR3⁺ cells in the granulocyte gate of cells isolated from blood of wild-type and STAT6^-/- mice following intratracheal transfer of OVA-specific in vitro polarized Th2 lymphocytes and 3 OVA or PBS challenges (n = 3 mice per group, from 1 experiment). E) Lung chemokine RNA copies normalized to copies of GAPDH RNA in wild-type and STAT6^-/- mice following intratracheal transfer of OVA-specific in vitro polarized Th2 lymphocytes and 3 OVA challenges (n = 6 mice per group, from 2 experiments).

Figure 4. STAT6 expression in myeloid derived cells in the lung does support Th2 cell and eosinophil trafficking into the airways

A) Number of OVA-specific Th2 cells in the BAL of wild-type BALB/c mice after bone marrow transplant with wild-type bone marrow or STAT6^-/- mice after bone marrow transplant with wild-type, STAT6^-/- or RAG1^-/- bone marrow, and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 4-8 mice per group, from 3 experiments). B) Number of CD4⁺ T cells, and CD8⁺ T cells in the BAL of STAT6^-/- mice after bone marrow transplant with wild-type, STAT6^-/-, or RAG1^-/- bone marrow, and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 2 experiments). C) Percentage and D) number of eosinophils in the BAL of STAT6^-/- mice after bone marrow transplant with wild-type, STAT6^-/- or RAG1^-/- bone marrow, and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 2 experiments). E) Representative lung histology from STAT6^-/- mice after bone marrow transplant with wild-type (i, iv - 100x and 400x respectively), STAT6^-/- (ii, v), or RAG1^-/- (iii, vi) bone marrow, and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 2 experiments). Black bars are 100 μm. F) Lung chemokine RNA copies normalized to copies of GAPDH RNA and G) BAL chemokine protein levels in STAT6^-/- mice after bone marrow transplant with wild-type, STAT6^-/- or RAG1^-/- bone marrow, and following transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 2 experiments).

Figure 5. DT administration selectively depletes myeloid dendritic cells in CD11b-DTR mice

A) Chemokine RNA copies normalized to copies of GAPDH from CD11c⁺/CD11b⁺ and CD11c⁺/CD11b^- cells sorted from the lungs of wild-type naïve mice and wild-type mice following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 4 pooled mice per group, from 1 experiment). B) Representative flow cytometry of lung cells isolated from CD11b-DTR mice 4 days after 2 treatments with PBS or DT. Cells are stained with labeled antibodies to CD11b and CD11c and gated on the monocytes/macrophages. C) Representative flow cytometry of lung cells isolated from CD11b-DTR mice 4 days after 2 treatments with PBS or DT. Cells were stained with labeled antibodies to Gr-1 (RB6-8C5 from BD Pharmingen), MHC II, CD11b and CD11c and gated on monocytes/macrophages and on CD11b⁺/CD11c⁺ cells. D) Number of CD11c⁺/CD11b⁺/MHC-II⁺/Gr-1^- cells in the lungs of CD11b-DTR mice as measured by flow cytometry after treatment with PBS or DT (n = 6 mice per group, from 2 experiments). E) Representative lung histology from CD11b-DTR mice 4 days after 2 treatments with PBS (i) or DT (ii). Lung sections are stained with hematoxylin and eosin. Black bar is 100 μm. F) Representative immunohistochemistry for GFP in lungs taken from CD11b-DTR mice 4 days after 2 treatments with PBS (i) or DT (ii).

Figure 6. DT administration to CD11b-DTR mice impairs Th2 cell and eosinophil recruitment into the airways in an adoptive transfer model of allergic airway inflammation

A) Number of OVA-specific Th2 cells and B) CD4⁺ and CD8⁺ T cells in the BAL of CD11b-DTR mice after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 3 experiments). C) Percentage and D) number of eosinophils in the BAL of CD11b-DTR mice after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 3 experiments). E) Lung chemokine RNA copies normalized to copies of GAPDH RNA in CD11b-DTR mice after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA or PBS challenges (n = 3-6 mice per group, from 2 experiments). F) Representative lung histology from CD11b-DTR mice treated with PBS (i) or DT (ii), and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 3 experiments). Black bars are 100 μm. G) Representative immunohistochemistry for GFP in lungs taken from CD11b-DTR mice after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 4 mice per group, from 1 experiment). Black bars are 10 μm. H) Number of CD11c⁺/CD11b⁺ cells in the lungs of CD11b-DTR mice as measured by flow cytometry after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 3 experiments).

Figure 7. Intratracheal transfer of bone marrow-derived dendritic cells restores Th2 cell recruitment in CD11b⁺ myeloid cell depleted CD11b-DTR mice

A) Bone marrow cells from wild-type or STAT6^-/- mice were cultured for 10 days in media supplemented with GM-CSF. The non-adherent cells were harvested and stained with antibodies to CD11b, CD11c, Gr-1, and MHC II. B) Number of OVA-specific Th2 cells in the BAL following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges in CD11b-DTR mice which had been injected with DT and had received intratracheal transfer of wild-type or STAT6^-/- bone marrow derived dendritic cells (n = 10 mice per group, from 2 experiments). CD11b-DTR mice treated with DT or PBS following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 8 mice per group, from 2 experiments) served as controls.

Figure 8. DT administration to CD11b-DTR mice does not impair Th2 cell accumulation in the lymph node but does reduce IL-13 induced chemokine production

A) Numbers of OVA-specific Th2 cells in the thoracic lymph nodes of CD11b-DTR mice after treatment with PBS or DT and following adoptive transfer of OVA-specific in vitro polarized Th2 lymphocytes and 4 OVA challenges (n = 5 mice per group, from 2 experiments). B) Lung chemokine RNA copies normalized to copies of GAPDH RNA in CD11b-DTR mice after treatment with PBS or DT and 24 hours following intranasal IL-13 administration (n = 6 mice per group, from 2 experiments).