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. 2021 Sep 13;22(18):9898.
doi: 10.3390/ijms22189898.

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury

Vincent Yi-Fong Su  1   2 , Wei-Chih Chen  1   3 , Wen-Kuang Yu  1   3 , Huai-Hsuan Wu  3 , Hao Chen  3 , Kuang-Yao Yang  1   3   4   5
Affiliations

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury

Vincent Yi-Fong Su et al. Int J Mol Sci. .

Abstract

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Keywords: CXCR2; GRK2; acute lung injury; chemotaxis; neutrophil chemotaxis; nintedanib; p38 MAPK.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Oral nintedanib (Nin) administration improved histological features and fibrosis in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). (A) Hematoxylin and eosin staining revealed minimal histopathological abnormalities in control mice injected intratracheally with phosphate-buffered saline (PBS). Oral nintedanib administration attenuated pathological changes in lung injury in mice with LPS-induced ALI. Mice with ALI treated with nintedanib had significantly lower lung injury scores than did mice with untreated ALI. (B) Immunohistochemical staining showed that intratracheal injection of LPS significantly increased the expression of collagen-1. The oral administration of nintedanib inhibited the fibrotic changes in mice with LPS-induced ALI. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. Nin (−); n = 6 per group.
Figure 2
Figure 2
Nintedanib (Nin) administration restored the expression of the neutrophil marker Ly6G, VLA-4 and VCAM-1 in the lungs of mice with LPS-induced ALI. (A–C) Intratracheal injection of LPS significantly increased the expression of Ly6G, VLA-4, and VCAM-1 in the lungs of mice with ALI compared with control mice. Nintedanib administration reversed these changes. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline; IHC, immunohistochemical stains.
Figure 2
Figure 2
Nintedanib (Nin) administration restored the expression of the neutrophil marker Ly6G, VLA-4 and VCAM-1 in the lungs of mice with LPS-induced ALI. (A–C) Intratracheal injection of LPS significantly increased the expression of Ly6G, VLA-4, and VCAM-1 in the lungs of mice with ALI compared with control mice. Nintedanib administration reversed these changes. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline; IHC, immunohistochemical stains.
Figure 3
Figure 3
Nintedanib (Nin) administration restored the phosphorylated-p38:p38 ratio (P-p38/p38) to inhibit neutrophil migration and fibrotic changes in mice with LPS-induced ALI. Western blots confirmed that the intratracheal injection of LPS significantly increased the expression of VLA-4 and VCAM-1 and collagen-1, and P-p38/p38, in the lungs of these mice. Oral nintedanib administration reversed these changes. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline.
Figure 4
Figure 4
Nintedanib (Nin) administration improved pulmonary polymorphonuclear neutrophil (PMN) recruitment in mice with acute lung injury (ALI). (A) The bronchoalveolar lavage fluid (BALF) showed marked accumulation of PMNs in the lungs of mice with LPS-induced ALI compared with control mice. Mice with ALI treated with nintedanib had significantly reduced PMN counts. (B) Immunofluorescence (IF) staining showed LPS induced the expression of Ly6G and VLA-4 on neutrophils in the BALF, and nintedanib downregulated this expression. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline.
Figure 5
Figure 5
Nintedanib (Nin) administration restored the changes in chemokine (C-X-C motif) receptor 2 (CXCR2) and G protein-coupled receptor kinase 2 (GRK2) expression on circulating neutrophils and prevented pulmonary neutrophil accumulation in mice with LPS-induced ALI. LPS reduced the expression of GRK2 and induced the expression of CXCR2 on circulating neutrophils in mice with LPS-induced ALI, and nintedanib administration restored these changes. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline.
Figure 6
Figure 6
Nintedanib (Nin) reduced human neutrophil migration stimulated by MIP-2 or LPS in a neutrophil migration model. Human neutrophils (2 × 105) isolated from patients with septic shock were placed in upper wells. MIP-2 or LPS enhanced human neutrophil migration. Nintedanib administration reversed these changes. Data are presented as the mean ± standard deviation. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline; HPF, high-power field.
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