β2 integrins rather than β1 integrins mediate Alternaria-induced group 2 innate lymphoid cell trafficking to the lung

Abstract

Background: Group 2 innate lymphoid cells (ILC2s) expand in the lungs of mice during type 2 inflammation induced by the fungal allergen Alternaria alternata. The increase in ILC2 numbers in the lung has been largely attributed to local proliferation and whether ILC2s migrate from the circulation to the lung after Alternaria exposure is unknown.

Objective: We examined whether human (lung, lymph node, and blood) and mouse lung ILC2s express β₁ and β₂ integrin adhesion molecules and whether these integrins are required for trafficking of ILC2s into the lungs of mice.

Methods: Human and mouse ILC2s were assessed for surface expression of β₁ and β₂ integrin adhesion molecules by using flow cytometry. The role of β₁ and β₂ integrins in ILC2 trafficking to the lungs was assessed by in vivo blocking of these integrins before airway exposure to Alternaria in mice.

Results: Both human and mouse lung ILC2s express high levels of β₁ and β₂ integrin adhesion receptors. Intranasal administration of Alternaria challenge reduced ILC2 numbers in the bone marrow and concurrently increased blood and lung ILC2 numbers. In vivo blocking of β₂ integrins (CD18) significantly reduced ILC2 numbers in the lungs but did not alter ILC2 proliferation, apoptosis, and function. In contrast, in vivo blocking of β₁ integrins or α₄ integrins did not affect lung ILC2 numbers.

Conclusion: ILC2 numbers increase in the mouse lung not only through local proliferation but also through trafficking from the circulation into the lung using β₂ rather than β₁ or α₄ integrins.

Keywords: Alternaria alternata; Group 2 innate lymphoid cell; adhesion; integrin.

Figure 1. Human ILC2s express CD18/CD11a (β₂/α_L) and CD29/CD49d (β₁/α₄) integrins

(A) Representative ILC2 gating strategy from human lymph nodes. (B) The frequency of ILC2s from CD45⁺ human PBMCs, lungs, and lung lymph nodes. Summary of ILC2 adhesion molecule expression from (C) PBMCs (n=7), (D) lung tissue (n=8), and (E) lymph nodes (n=7). Comparison of ILC2 and CD4⁺CRTH2⁺ cell adhesion molecule ΔgMFI from (F) PBMCs (n=5), (G) lungs (n=3), and (H) lymph nodes (n=5) of the same donor. Solid circles represent ILC2 populations and open circles represent CD4⁺CRTH2⁺ populations. Data depicted as boxplots (whiskers: 10^th–90^th percentile) or individual points; mean represented as a line ± SEM. *P<0.05 by Student’s paired t test.

Figure 2. Adhesion molecule expression on mouse lung ILC2s

(A) Representative gating strategy of mouse lung ILC2s. Mouse lung ILC2s (open circles) compared to lung CD4⁺Thy1.2⁺ cells (gray squares) (B) percent adhesion molecule expression and (C) adhesion molecule ΔgMFI (n=3).Comparison of mouse lung ILC2 (D) percent expression and (E) ΔgMFI of adhesion molecules between naïve mice (open circles) and Alternaria challenged mice (solid circles; n=7). Independent experiments depicted as individual points; mean represented as a line ± SEM. *P<0.05, **P<0.01, ***P<0.001 by Student’s t test.

Figure 3. ILC2 levels change in bone marrow, blood, and lungs following Alternaria challenge in wt and BMT mice

Total ILC2s from (A) bone marrow, (B) blood, and (C) whole lung from naïve (white; n≥23), 4-day Alternaria challenged (gray; n=8), and 7-day Alternaria challenged mice (black; n≥14). CD45.1 or CD45.2 donor bone marrow was transplanted into CD45.2 irradiated recipient mice (D) Representative plots of BMT mouse blood ILC2 expression of CD45.1 and CD45.2. (E) Total CD45.1 (black) and CD45.2 (white) blood cells from BMT mice. Total ILC2s from (F) blood and (G) whole lung from naïve (n=8) or Alternaria challenged (n=4) BMT mice. (H) Total lung Ki-67⁺ ILC2s from BMT mice. Data summarized as bar graphs representing mean ± SEM. *P< 0.05, **P< 0.01, ***P< 0.001 by Student’s t test.

Figure 4. In vivo blocking of CD18 diminishes Alternaria-induced ILC2 recruitment to the airways

(A) Mice treated with anti-CD18 antibody (αCD18) or the isotype control and challenged with Alternaria (Alt) (B) Total ILC2s per mouse whole lung (n≥10). (C) Representative plots of ILC2 Ki-67 and Annexin V expression and summary of the percentage of Ki-67⁺ ILC2s (n≥10) and live Annexin V⁺ ILC2s (n≥4) per mouse lung. (D) Summary of total IL-5, IL-13, and IL-5/IL-13 producing ILC2s in the lung (n≥4; data was log transformed for normalcy). (E) Total number of sorted ILC2s adherent to ICAM-1 coated wells compared to control coated wells ± preincubation of ILC2s with αCD18 (two independent experiments). (F) Total ILC2s per lung of mice treated ± Alt and ± anti-CD11a antibody (n≥6). Data summarized as bar graphs or individual points; mean represented as a line ± SEM. (NS) not significant, *P< 0.05, **P< 0.01, ***P< 0.001 by Student’s t test.

Figure 5. In vivo blocking of CD29 and CD49d does not alter Alternaria–induced ILC2 recruitment

(A) Total number of lung ILC2s, (B) total Ki-67⁺ ILC2s, and (C) live Annexin V⁺ ILC2s per mouse treated ± anti-CD29 antibody (n≥6). (D) Percentage of human ILC2s expressing integrin β₇ and both CD49d and β₇ from PBMCs (white; n=4), lung tissue (black; n=3), and lymph nodes (gray; n=4). (E) Summary of the percentage of mouse lung ILC2s expressing CD49d and integrin β₇ from both naïve and Alt challenged mice (n=8). (F) Total number of lung ILC2s, (G) total Ki-67⁺ ILC2s, and (H) live Annexin V⁺ ILC2s per mouse treated ± anti-CD49d antibody (n≥5). Mean represented as a line ± SEM. (NS) not significant, *P< 0.05, **P< 0.01, ***P< 0.001 by Student’s t test.