Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome
Lisa R. Young, Peter M. Gulleman, Chelsi W. Short, Harikrishna Tanjore, Taylor Sherrill, Aidong Qi, Andrew P. McBride, Rinat Zaynagetdinov, John T. Benjamin, William E. Lawson, Sergey V. Novitskiy, Timothy S. Blackwell
Lisa R. Young, Peter M. Gulleman, Chelsi W. Short, Harikrishna Tanjore, Taylor Sherrill, Aidong Qi, Andrew P. McBride, Rinat Zaynagetdinov, John T. Benjamin, William E. Lawson, Sergey V. Novitskiy, Timothy S. Blackwell
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Research Article Pulmonology

Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

Abstract

Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-β production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-β in myeloid cells or of TGF-β signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-β, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.

Authors

Lisa R. Young, Peter M. Gulleman, Chelsi W. Short, Harikrishna Tanjore, Taylor Sherrill, Aidong Qi, Andrew P. McBride, Rinat Zaynagetdinov, John T. Benjamin, William E. Lawson, Sergey V. Novitskiy, Timothy S. Blackwell

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Figure 6

Macrophage TGF-β contributes to fibrotic susceptibility in HPS1 mice.

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Macrophage TGF-β contributes to fibrotic susceptibility in HPS1 mice. (A...
(A–C) Mice deficient in myeloid TGF-β1 (LysM.Cre/TGFb1f/f; denoted WT/TGF-βΔMye) were studied in comparison to LysM.Cre– littermate controls (LysM.Cre–/TGFb1f/f; denoted WT). (A) Macrophages were isolated by bronchoalveolar lavage (BAL), and total TGF-β1 was measured in the cell culture media by ELISA after 24 hours in culture (mean ± SEM); n = 3/group; *P < 0.05 by Mann-Whitney U analysis. (B) BAL cell counts from unchallenged mice; n = 5 WT and n = 7 WT/ TGF-βΔMye, P = NS by Mann-Whitney U analysis. (C) Total TGF-β1 in lung homogenates from unchallenged mice. Data are presented as box-and-whisker Tukey plots; n = 4/group, P = NS by Mann-Whitney U analysis. (D–H) TGF-βΔMye mice were bred onto the HPS1 background to generate HPS1/TGF-βΔMye mice or HPS1/Cre– littermate controls (denoted HPS1). (D) Total TGF-β1 was determined by ELISA in BAL fluid from unchallenged mice; n = 15 WT/TGF-βΔMye, n = 8 HPS1/TGF-βΔMye, n = 7 WT controls, and n = 11 HPS1 controls. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/TGF-βΔMye. (E) Representative H&E histologic images (original magnification, ×10) of lung sections at 7 days after IT bleomycin. (F) Lung collagen content of the left lung in unchallenged mice and at 7 days after bleomycin (mean ± SEM). For unchallenged groups, n = 6. For bleomycin-challenged groups, n = 3 WT, n = 6 WT/TGF-βΔMye, n = 21 HPS1, and n = 15 HPS1/TGF-βΔMye mice. Comparisons between bleomycin-challenged groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. other bleomycin-challenged groups. (G) Fibrosis scoring on trichrome-stained lung tissue (mean ± SEM); n = 3/group for WT and n = 6/group for HPS1, *P < 0.05. (H) TUNEL+ alveolar epithelial cells in lung sections from mice 24 hours after bleomycin challenge (mean ± SEM); n = 10 WT/LysM.Cre– (WT) and n = 12 for other groups. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/ TGF-βΔMye.