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 2

CCR2 regulates increased recruitment of macrophages and exaggerated fibrotic susceptibility in HPS mice.

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CCR2 regulates increased recruitment of macrophages and exaggerated fibr...
HPS1 or HPS2 mutant mice were bred to homozygosity with global CCR2-deficient (CCR2–/–) mice. (A) Numbers of macrophages in unchallenged and bleomycin-treated mice (day 7) were quantified by flow cytometry using the F4/80 marker. Data are presented as box-and-whisker Tukey plots. For unchallenged groups, n = 13 WT, n = 9 WT/CCR2–/–, n = 6 HPS1, n = 4 HPS1/CCR2–/–, n = 4 HPS2, and n = 10 HPS2/CCR2–/–. For bleomycin-challenged groups, n = 7 WT and WT/CCR2–/–, n = 4 HPS1 and HPS1/CCR2–/–, n = 10 HPS2, and n = 6 HPS2/CCR2–/–. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.05 vs. unchallenged WT and CCR2–/– groups, **P < 0.05 vs. bleomycin-challenged WT and CCR2–/– groups. (B) Survival after IT bleomycin; n = 8/group WT and WT/CCR2–/–, n = 14 HPS2, and n = 10 HPS2/CCR2–/–. P < 0.05 for HPS2/CCR2–/– vs. other groups by log-rank test. (C) Histology of lung sections from mice 7 days after bleomycin challenge. Representative H&E images; original magnification, ×10. (D) Lung collagen content of left lung quantified by Sircol assay in unchallenged mice or 7 days after bleomycin challenge (mean ± SEM). For unchallenged groups, n = 4 WT and n = 6 WT/CCR2–/–. For bleomycin-challenged groups, n = 11 WT, n = 10 WT/CCR2–/–, n = 17 HPS1, n = 19 HPS1/CCR2–/–, n = 24 HPS2, and n = 15 HPS2/CCR2–/–. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.001 for HPS2/CCR2+/+ and HPS1/CCR2+/+ vs. WT and CCR2–/– groups. (E) Modified Ashcroft fibrosis score from trichrome-stained lung sections. Data are presented as box-and-whisker Tukey plots; n = 4/group, except n = 5 HPS1/CCR2–/– and HPS2, and n = 6 HPS2/CCR2–/–, with comparisons between groups conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT and CCR2–/– groups. (F) TUNEL+ alveolar epithelial cells (AECs) in lung sections from mice 24 hours after bleomycin challenge; n = 6 HPS1, n = 5 HPS2, n = 10 HPS1/CCR2–/–, and n = 7 HPS2/CCR2–/–. Comparison with CCR2–/– groups was assessed using Mann-Whitney U analysis, *P < 0.01.