Alveolar macrophages are sentinels of murine pulmonary homeostasis following inhaled antigen challenge

Abstract

Background: Alveolar macrophages are sentinels of the pulmonary mucosa and central to maintaining immunological homeostasis. However, their role in governing the response to allergen is not fully understood. Inappropriate responses to the inhaled environment manifest as asthma.

Methods: We utilized a mechanistic IL-13-driven model and a house dust mite allergen mucosal sensitization model of allergic airway disease to investigate the role of alveolar macrophages in regulating pulmonary inflammation.

Results: IL-13-dependent eosinophilic and Th2 inflammation was enhanced in mice depleted of alveolar macrophages using clodronate liposomes. Similarly, depletion of alveolar macrophages during house dust mite sensitization or established disease resulted in augmented Th2 immunity and increased allergen-specific IgG1 and IgE. Clodronate treatment also delayed the resolution of tissue inflammation following cessation of allergen challenge. Strikingly, tissue interstitial macrophages were elevated in alveolar macrophage-deficient mice identifying a new homeostatic relationship between different macrophage subtypes. A novel role for the macrophage-derived immunoregulatory cytokine IL-27 was identified in modulating Th2 inflammation following mucosal allergen exposure.

Conclusions: In summary, alveolar macrophages are critical regulators of Th2 immunity and their dysregulation promotes an inflammatory environment with exacerbation of allergen-induced airway pathology. Manipulating IL-27 may provide a novel therapeutic strategy for the treatment of asthma.

Keywords: Alveolar macrophage; homeostasis; house dust mite; interleukin-13; lung.

Figure 1

IL-13-induced Th2 airway pathology is amplified following the depletion of alveolar macrophages (A) Schematic depicting either PBS or clodronate (Clod) liposome and IL-13 administration protocol. Mice received either clodronate-containing liposomes (Clod) or PBS-containing liposomes (PBS). 48 hours later, mice received either 5 μg of IL-13 or PBS + 1% FCS (vehicle) for 3 consecutive days. Mice were culled 15 h later. (B) alveolar macrophages (C) total airway leukocytes (D) eosinophils (E) neutrophils (F) Th2 lymphocytes retrieved following broncho-alveolar lavage and quantified by flow cytometry. BAL fluid was analyzed for inflammatory cytokines by ELISA (G) IL-4 and (H) IL-5. *P < 0.05 and **P < 0.01 relative to PBS control group by Mann–Whitney U-test, n = 6–8.

Figure 2

The absence of alveolar macrophages during mucosal allergen sensitization and challenge augments house dust mite-induced Th2 inflammation in the airways (A) Schematic depicting either PBS or clodronate (Clod) liposome and house dust mite (HDM) administration. (B) Total airway leukocytes, (C) eosinophils, (D) neutrophils, and (E) Th2 lymphocytes retrieved following bronchoalveolar lavage and quantified by flow cytometry. BAL fluid was analyzed for inflammatory cytokines by ELISA (F) IL-4, (G) IL-5, and (H) IL-10. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to PBS control group by Mann–Whitney U-test, n = 6–8.

Figure 3

Disruption of alveolar macrophage-directed homeostasis promotes an increase in interstitial lung tissue macrophages and enhances the systemic immune response following HDM mucosal challenge (A) Total tissue leukocytes and (B) lung interstitial macrophages quantified by flow cytometry. (C) FACS plots depicting lung alveolar and interstitial macrophage populations. (D) Total IgE, (E) HDM-specific IgE, (F) HDM-specific IgG1, and (G) HDM-specific IgG2a in serum following clodronate treatment during sensitization quantified by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to PBS control group by Mann–Whitney U-test, n = 6–8.

Figure 4

Alveolar macrophages are elevated concomitant with the resolution of mucosal inflammation (A) Schematic depicting PBS or HDM, (B) total tissue leukocytes, (C) eosinophils, and (D) neutrophils were quantified by differential counting. (E) Th2 lymphocytes quantified by flow cytometry. Th2 cytokine levels in lung tissue homogenate were quantified by ELISA (F) IL-5 and (G) IL-13. (H) LC/MS/MS was used to quantify lung tissue lipoxin A4. (I) CD4⁺CD25⁺FoxP3⁺ T-regulatory lymphocytes and (J) alveolar macrophages were quantified by flow cytometry. At each time point n = 4–12 mice/group. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to PBS control group by Mann–Whitney U-test. ⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001 relative to peak inflammation, 4-h time point group by Mann–Whitney U-test.

Figure 5

The return to homeostasis after allergen challenge is disrupted following the removal of alveolar macrophages (A) Schematic depicting PBS or clodronate (Clod) liposome administration protocol following the induction of allergic airways disease. (B) airway leukocytes retrieved following broncho-alveolar lavage and (C) tissue eosinophils; (D) tissue neutrophils determined by differential counting. (E) Lung tissue Th2 lymphocytes quantified by flow cytometry. (F) lung tissue chemokines measured by ELISA. (G) interstitial macrophages determined by flow cytometry, n = 3–6 mice/group, bar represents median. *P < 0.05 and **P < 0.01 relative to PBS control group by Mann–Whitney U-test, n = 3–6.

Figure 6

Exacerbation of IL-13- and HDM-induced disease following alveolar macrophage depletion is associated with reduced levels of the immuno-regulatory cytokine IL-27. IL-27 mRNA expression in lung FACS-sorted macrophages analyzed by qPCR (A) F4/80⁺ CD11c+ alveolar macrophages, (B) IL-13-treated mice, and (C) HDM-treated mice with alveolar macrophage depletion during sensitization and during resolution *P < 0.05 and **P < 0.01 relative to PBS control group by Mann–Whitney U-test, n = 5–8.