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. 2022 Dec 13;41(11):111797.
doi: 10.1016/j.celrep.2022.111797.

Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis

Hasan H Öz  1 , Ee-Chun Cheng  1 , Caterina Di Pietro  1 , Toma Tebaldi  2 , Giulia Biancon  3 , Caroline Zeiss  4 , Ping-Xia Zhang  5 , Pamela H Huang  1 , Sofia S Esquibies  1 , Clemente J Britto  6 , Jonas C Schupp  7 , Thomas S Murray  1 , Stephanie Halene  3 , Diane S Krause  8 , Marie E Egan  9 , Emanuela M Bruscia  10
Affiliations

Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis

Hasan H Öz et al. Cell Rep. .

Abstract

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor β (TGF-β) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-β signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.

Keywords: CP: Immunology; c-c motif chemokine receptor 2; chronic lung inflammation; cystic fibrosis; lipopolysaccharide; lung remodeling; macrophages; monocytes; neutrophils; recruitment; transforming growth factor β.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. CCR2+ monocytes and monocyte-derived macrophages are abundant in CF lungs
(A) Representative CCR2 immunohistochemistry and H&E staining in human lung tissue from healthy donors (non-CF, a–c) and patients with CF (CF, d–f) (n = 3). Greater magnification of c and f is shown in g and h, respectively. B, bronchiole. Magnification for a–f, 100×; magnification for g and h, 400×. Scale bars: 500 μm (a–f) and 100 μm (g and h). (B-D) scRNA-seq data from cells isolated from induced sputum of healthy individuals (CTRL) versus patients with CF. (B) Uniform manifold approximation and projection (UMAP) visualization of 20,095 sputum cells from nine patients with CF and five control (CTRL) subjects. Each dot represents a single cell, and cells are labeled by (B) cell type (AM, alveolar macrophage; B, B cell; DC, dendritic cell; Epithelia, epithelial cell; M, macrophage; Mono, monocyte; PMN, polymorphonuclear neutrophil granulocyte; pDC, plasmacytoid DC; T/NK, T and NK cells), (C) disease state (CTRL versus CF cells), and (D) expression of at least one CCR2 mRNA molecule (red). Marker gene expression to identify cell populations is shown in Figure S1C. (E) Boxplot of the percentage of CCR2-expressing cells in the monocyte population (CTRL versus CF). See also Figure S1 and Table S1.
Figure 2.
Figure 2.. Elevated numbers of cMons and monocyte-derived macrophages promote and perpetuate neutrophilic pulmonary inflammation in CF
(A) Chronic LPS model. WT, CF, dKO, and CCR2−/− mice were nebulized with LPS from PA 3 times per week over 5 weeks (15 doses total) and sacrificed 24 h after the last nebulization (chronic endpoint [T1]) or left to recover for 6 weeks (recovery endpoint [T2]). At each time point, BALF and lung tissue were collected for flow cytometry, histology, RNA, and protein analysis. T0 indicates untreated mice. (B) Total cell numbers of total AMs, moAMs, trAMs, IMs, cMons, Ly6C− Monos, and neutrophils were quantified by flow cytometry as percentage of viable cells multiplied by the total cell count in the inferior lung lobe. The gating strategy is described in Figure S3. Data are generated from three independent experiments with 2–6 mice per genotype and time point. Each biological replicate is represented by a dot. Bars are depicted as means ± SEM, and p values were calculated using one-way ANOVA and Tukey’s test for multiple comparisons between the genotypes for each time point separately (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figures S2 and S3.
Figure 3.
Figure 3.. Enhanced CCR2-mediated monocyte recruitment drives pathogenic tissue remodeling in CF lungs
(A and B) Representative subgross H&E images of lung from WT, CF, dKO, and CCR2−/− mice at T0, T1, and T2. Black stars indicate enlarged alveolar spaces. Scale bars: 500 μm (A) and 50 μm (B). (C) Relative alveolar disruption score from semiquantification analysis of lung histology. Bars are depicted as means ± SEM for 4 or more mice per genotype and time point. (D) Relative weight loss of mice after week 1 of LPS administration. Each dot represents one biological replicate. r multiple comparisons between the genotypes for each time point separately (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figures S4, S5, and S6.
Figure 4.
Figure 4.. CCR2-derived lung monocytes/macrophages drive pathogenic TGF-β signaling in CF lungs
(A) Quantification of active (left) and total (right) TGF-β protein in BALF by ELISA. (B and D) The mRNA expression levels of Tgfβ (B) and TGF-β signaling target genes (D) in lung tissues of WT, CF, dKO, and CCR2−/− mice at T0, T1, and T2. The average expression from two technical replicates was normalized to 18S and WT at T0. (C) Densitometric analysis of Western blot for SMAD2 and pSMAD2 from lung tissues of WT, CF, dKO, and CCR2−/− mice at T0, T1, and T2. The expression was normalized to VINCULIN and the WT at T0. Data are generated from three independent experiments with 2–4 mice per genotype and time point. Each biological replicate is represented by a dot. Bars are depicted as means ± SEM, and p values were calculated using one-way ANOVA and Tukey’s test for multiple comparisons between the genotypes for each time point separately (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S7.
Figure 5.
Figure 5.. cMons in CF perpetuate immune activation and neutrophil recruitment
(A) MDS plot based on RNA expression profiles of cMons, IMs, moAMs, and trAMs at T1 and T2. (B) The number of differentially expressed genes (DEGs) when comparing CF with the WT in each cell population at T1 or T2. (C) Pathway analysis of genes differentially expressed in CF versus WT cMons at T2. The plot shows the number of up- or downregulated genes associated with each pathway. Two samples per genotype and time point were used for analysis. (D) Schematic of the differential analysis depicted in (E). (E) Numbers of differentially up- or downregulated genes between T1 and T2 in each population. (F) Gene Ontology (GO) biological process enrichment analysis of differentially regulated genes in CF between T1 and T2 compared with the WT for each population (cMons, IMs, moAMs, and trAMs). Node size: odds ratio associated with enrichment; p value for Fisher’s exact test. Data were generated from 2 samples per genotype and time point. To ensure sufficient cell counts at T2, lungs from 2 WT or 2 CF mice were pooled for one sample. See also Figures S9, S10, and S11.
Figure 6.
Figure 6.. CFTR deficiency in CCR2-expressing cells drives excessive accumulation of lung immune cells in response to chronic LPS
(A) Schematic of the chronic LPS model and Ccr2cre/+Cftrfl/fl mice. (B) Total cell numbers of AMs, moAMs, trAMs, IMs, cMons, Ly6C− Monos, neutrophils, DCs, CD4 T cells, CD8 T cells, and B cells in lung tissue homogenates were quantified by flow cytometry as percentage of viable cells multiplied by the total cell count in the inferior lung lobe. The gating strategy is described in Figure S3. (C) Quantification of active and total TGF-β levels in BALF at T1 by ELISA. (D) Weight loss during 5 weeks of chronic LPS (right) and after the first week of LPS (left). (E) CBCs at T0 and T1. Data are generated from two independent experiments with 2–3 mice per genotype and time point. Each biological replicate is represented by a dot. Bars are depicted as means ± SEM, and p values were calculated using one-way ANOVA and Tukey’s test for multiple comparisons between the genotypes for each time point separately (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 7.
Figure 7.. Pharmacological targeting of CCR2 mitigates cMon recruitment and normalizes TGF-β levels in CF lungs
(A) Chronic infection model. CF mice were treated with CCR2 inhibitor (CCR2inh) starting 3 days before the first nebulization and throughout the experiment. (B) Quantification of flow cytometry analysis of lung immune cells (see Figures 1B and 1C) of CF + CCR2inh-treated mice compared with WT and CF mice at T1. (C) Active levels of TGF-β at T1. (D) Densitometric analysis of western blot bands from lung tissues of WT, CF, and CF + CCR2inh mice at T1. The expression levels were normalized to VINCULIN and the WT at T0. (E) mRNA expression levels in lung tissues of WT, CF, and CF + CCR2inh mice at T1. The expression was normalized to 18S and the WT at T0. Each biological replicate per genotype and experimental condition is represented by a dot. Bars are depicted as means ± SEM, and p values were calculated using one-way ANOVA and Tukey’s test for multiple comparisons between the genotypes for each time point separately (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figure S12.
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