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. 2024 Jun 22;25(1):257.
doi: 10.1186/s12931-024-02884-1.

Deregulated immune cell recruitment orchestrated by c-MET impairs pulmonary inflammation and fibrosis

Catarina Barbosa-Matos  1   2 , Caroline Borges-Pereira  1   2 , Sofia Libório-Ramos  1   2 , Raquel Fernandes  1   2 , Marcela Oliveira  1   2 , Ana Mendes-Frias  1   2 , Ricardo Silvestre  1   2 , Nuno S Osório  1   2 , Hélder N Bastos  3   4   5 , Rita F Santos  4   6 , Susana Guimarães  7 , António Morais  3 , Massimiliano Mazzone  8   9 , Agostinho Carvalho  1   2 , Cristina Cunha  1   2 , Sandra Costa  10   11
Affiliations

Deregulated immune cell recruitment orchestrated by c-MET impairs pulmonary inflammation and fibrosis

Catarina Barbosa-Matos et al. Respir Res. .

Abstract

Background: Pulmonary fibrosis (PF) represents the pathologic end stage of several interstitial lung diseases (ILDs) associated with high morbidity and mortality rates. However, current treatments can only delay disease progression rather than provide a cure. The role of inflammation in PF progression is well-established, but new insights into immune regulation are fundamental for developing more efficient therapies. c-MET signaling has been implicated in the migratory capacity and effector functions of immune cells. Nevertheless, the role of this signaling pathway in the context of PF-associated lung diseases remains unexplored.

Methods: To determine the influence of c-MET in immune cells in the progression of pulmonary fibrosis, we used a conditional deletion of c-Met in immune cells. To induce pulmonary fibrosis mice were administered with bleomycin (BLM) intratracheally. Over the course of 21 days, mice were assessed for weight change, and after euthanasia at different timepoints, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. Furthermore, c-MET expression was assessed in cryobiopsy sections, bronchoalveolar lavage fluid cells samples and single cell RNA-sequencing dataset from human patients with distinct interstitial lung diseases.

Results: c-MET expression was induced in lung immune cells, specifically in T cells, interstitial macrophages, and neutrophils, during the inflammatory phase of BLM-induced PF mouse model. Deletion of c-Met in immune cells correlated with earlier weight recovery and improved survival of BLM-treated mice. Moreover, the deletion of c-Met in immune cells was associated with early recruitment of the immune cell populations, normally found to express c-MET, leading to a subsequent attenuation of the cytotoxic and proinflammatory environment. Consequently, the less extensive inflammatory response, possibly coupled with tissue repair, culminated in less exacerbated fibrotic lesions. Furthermore, c-MET expression was up-regulated in lung T cells from patients with fibrosing ILD, suggesting a potential involvement of c-MET in the development of fibrosing disease.

Conclusions: These results highlight the critical contribution of c-MET signaling in immune cells to their enhanced uncontrolled recruitment and activation toward a proinflammatory and profibrotic phenotype, leading to the exacerbation of lung injury and consequent development of fibrosis.

Keywords: Bleomycin; ILDs; Inflammation; Pulmonary fibrosis; c-MET.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
c-MET expression is enhanced in immune cells during BLM-induced lung inflammation. (A) Mouse model of pulmonary fibrosis, induced by BLM intratracheal administration. This model comprises an inflammatory reaction which vanishes gradually and it is followed by the fibrotic phase. Several readouts were analyzed at specific time points throughout both phases of disease progression, which are mentioned in the scheme associated with the respective time point. Percentage of c-MET+ among (B) T cells, (C) interstitial macrophages, (D) neutrophils, (E) alveolar macrophages and (F) eosinophils, horizontal dashed line represents the mean of the vehicle group (day 0), and MFI of c-MET in lung (G) T cells, (H) interstitial macrophages and (I) neutrophils in the lungs of vehicle-administered mice (day 0) and throughout the inflammatory phase of BLM-induced PF (day 1, day 3, day 5 and day 7). n = 5–14 per group. Data are expressed as mean ± SEM and were analyzed with one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, comparison with vehicle group. #p < 0.05, ##p < 0.01, ###p < 0.001, comparison between time points. For all the experiments, similar results were obtained in three independent experiments
Fig. 2
Fig. 2
Deletion of c-Met from immune cells attenuates lung injury following BLM administration. WT and cKO mice administered intratracheally with BLM were followed for (A) weight change and (B) survival. n = 5–24 mice per group. (C) Representative images of H&E-stained lung sections from vehicle-administered mice, and on days 5 and 7 after BLM injection and (D) respective scoring. Original magnification × 20, scale bar 50 µm. LDH activity in the (E) dissociated tissue and (F) BAL supernatant at 7 days after BLM injection. (G) Gene expression of repair-associated molecules (Tgf-β1, Igf-1, Ctgf) in the lung at 7 days after BLM injection. n = 12–14 per group. Data are expressed as mean ± SEM and were analyzed by Student’s two-tailed t test (A, D, G), log-rank test (B) or with one-way ANOVA with Tukey’s multiple comparisons test (E, F). *p < 0.05, **p < 0.01. Similar results were obtained in three independent experiments
Fig. 3
Fig. 3
c-Met deletion in immune cells promotes early inflammatory cells recruitment after BLM administration. Number of (A) T cells, (B) interstitial macrophages, (C) neutrophils, (D) alveolar macrophages and (E) eosinophils. Cell numbers were calculated based on the total number of cells in the dissociated lung. n = 8–10 per group. Data are expressed as mean ± SEM. and were analyzed by Student’s two-tailed t test. *p < 0.05. Similar results were obtained in three independent experiments
Fig. 4
Fig. 4
Boosted leukocyte chemoattraction and restrained proinflammatory milieu in cKO mice lung mainly on day 5 following BLM administration. Heatmap of the concentration, pg/mL, of (A) chemokines and (B) proinflammatory cytokines in lung on day 3, 5 and 7 and (C) interleukins associated with inflammatory cells phenotypic activation in lung on day 5 after bleomycin administration. n = 6 per group. Pvalue for each comparation is reported, Student’s two-tailed t test
Fig. 5
Fig. 5
Attenuated lung fibrosis in cKO mice on day 14 following BLM administration. (A) Representative images of lung sections stained with H&E and Masson’s trichrome. Original magnification × 10, scale bar 100 µm. (B) Ashcroft scoring of lung fibrosis. (C) Hydroxyproline levels in lungs. (D) Gene expression of profibrotic mediators in whole-lung samples. n = 12–14 per group (A-C) or data representative of one of three independent experiments n = 6 per group (D). Data are expressed asmean ± SEM and were analyzed by Student’s two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. Similar results were obtained in three independent experiments
Fig. 6
Fig. 6
c-MET expression is augmented in T cells in fibrosis-related ILDs. (A) Representative images of immunofluorescence on c-MET (green) in T cells (red) and (B) respective expression quantification on human lung sections from a cohort of patients. Original magnification × 20, scale bar 50 µm. (C) Percentage of T cells with c-MET expression, quantified by flow cytometry on cryopreserved BAL samples from a cohort of patients. (D) Plot representing expression of c-MET in T cells from donor and ILD patients. n = 1–16 per group. Data are expressed as mean ± SEM and were analyzed with Kruskal–Wallis test with Dunn's multiple comparisons test. *p < 0.05, **p < 0.01
Fig. 7
Fig. 7
Schematic illustration of the importance of c-MET in immune cells during BLM-induced PF progression. Lung injury is induced by BLM leading to uncontrolled inflammatory recruitment, followed by excessive deposition of extracellular matrix proteins, as collagen. Absence of c-Met in immune cells resulted in anticipated inflammatory recruitment, attenuated proinflammatory environment, leading to higher epithelial repair, which later culminates in lower collagen deposition and prevents fibrosis
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