Nintedanib downregulates the profibrotic M2 phenotype in cultured monocyte-derived macrophages obtained from systemic sclerosis patients affected by interstitial lung disease

Abstract

Background: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.

Methods: Fourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients' informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFβ1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.

Results: Cultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).

Conclusions: In cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFβ1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

Keywords: Alternatively activated macrophages; Fibrosis; Interstitial lung disease; Systemic sclerosis; Tyrosine kinases inhibitor.

Fig. 1

Study design of in vitro experiments with cultured MDMs obtained from SSc-ILD patients. After collecting a venous blood sample and isolating peripheral blood mononuclear cells through density gradient centrifugation using Ficoll-Paque, human monocytes were extracted using an isolation kit and then stimulated to induce differentiation in monocyte-derived macrophages (MDMs). An aliquot of cultured MDMs isolated from each enrolled SSc-ILD patients was subsequently maintained in growth medium (untreated), another aliquot was treated with nintedanib at concentrations of 0.1µM and another aliquot was treated with nintedanib at the concentration of 1µM for 3, 16, 24 h. PBMCs: peripheral blood mononuclear cells; PMA: phorbol myristate acetate; MDMs: monocyte-derived macrophages. The picture was created with BioRender.com

Fig. 2

Western blotting with related densitometric analysis of the protein synthesis and gene expression of surface and functional markers of cultured MDMs from HS, SSc-ILD patients and SSc patients without ILD. (A) Evaluation by Western blotting and related densitometric analysis of the protein synthesis of CD204, CD206, CD163, and MerTK, and (B) evaluation by quantitative real time polymerase chain reaction (qRT-PCR) of the gene expression of MerTK and TGFβ1 in cultures of monocyte-derived macrophages (MDMs) obtained from 5 voluntary healthy subjects (HS), 4 SSc patients without ILD (SSc patients no-ILD, SSc pts no-ILD), and 10 SSc-ILD patients (SSc-ILD pts). Cultured MDMs were maintained in normal growth medium without any treatment for 24 h. The value of protein expression of CD204, CD206, CD163, and MerTK was normalized to that of the corresponding GAPDH in cultured HS-MDMs, MDMs from SSc pts no-ILD, and MDMs from SSc-ILD pts. The resulting value of the protein expression of each molecule in cultured MDMs from SSc-ILD pts and from SSc pts no-ILD was compared with that obtained in cultured HS-MDMs (taken as unit value). Gene expression of MerTK and TGFβ1 corresponds to the expression level (fold-increase) of the target gene in cultured MDMs from SSc pts no-ILD, and MDMs from SSc-ILD pts was compared with that obtained in cultured HS-MDMs (taken as unit value). Data are reported as median with range. The protein and gene expression of each molecule obtained in cultured MDMs from SSc pts no-ILD and SSc-ILD represent the fold increase compared to the unit value of cultured HS-MDMs. Data are reported as median with range of fold increase compared to HSs

Fig. 3

Gene expression of cell membrane and functional markers of profibrotic M2 phenotype in cultured MDMs obtained from SSc-ILD patients. Evaluation by quantitative real time polymerase chain reaction (qRT-PCR) of the gene expression of CD204, CD206, CD163, MerTK, IL10, and TGFβ1 in cultures of monocyte-derived macrophages (MDMs) obtained from 10 SSc-ILD patients. Cultured MDMs were maintained in normal growth medium without any treatment (black bar), treated with nintedanib at the concentration of 0.1µM (light gray bar), treated with nintedanib at the concentration of 1µM (dark gray bar) for 3, 16, and 24 h. Gene expression corresponds to the expression level (fold-increase) of the target gene of nintedanib-treated SSc macrophages compared with that of untreated cells, taken as the unit value. Data are reported as median with range of fold increase compared to untreated cells

Fig. 4

Western blotting and related densitometric analysis of the protein synthesis of surface and functional markers of cultured MDMs from SSc-ILD patients. Evaluation by Western blotting and related densitometric analysis of the protein synthesis of CD204, CD206, CD163, and MerTK in cultured monocyte-derived macrophages (MDMs) obtained from 10 SSc-ILD patients. Cultured MDMs were maintained in normal growth medium without any treatment (untreated cells, black bar), treated with nintedanib at the concentration of 0.1µM (light gray bar), treated with nintedanib at the concentration of 1µM (dark gray bar) for 3, 16, and 24 h. For each experimental condition, the value of protein synthesis of CD204, CD206, CD163, and MerTK was normalized to that of the corresponding GAPDH. The resulting value of each treatment was compared with that of the related untreated cells (taken as unit value). Data are reported as median with range of fold increase compared to untreated cells

Fig. 5

TGFβ1 protein synthesis of cultured MDMs from SSc-ILD patients. Evaluation by ELISA of the release of active TGFβ1 in conditioned medium of cultured monocytes-derived macrophages obtained from 10 SSc-ILD patients. Cultured MDMs were maintained in normal growth medium without any treatment (black bar), treated with nintedanib at the concentration of 0.1µM (light gray bar), treated with nintedanib at the concentration of 1µM (dark gray bar) for 3, 16, and 24 h. Data are reported as median with range