Prevention of bleomycin-induced pulmonary fibrosis by vaccination with the Tocilizumab mimotope

Abstract

Mimotope, a kind of peptide vaccine, is developed to bind natural receptor and inhibit the downstream signaling. We have demonstrated that the vaccination of Tocilizumab mimotopes could alleviate the renal fibrosis by interfering with both IL-6 and ferroptosis signaling. However, the effect of the vaccination of Tocilizumab mimotopes on the fibroblast was not investigated in previous study. Thus, we sought to explore the changes in the fibroblast induced by the Tocilizumab mimotopes vaccination. Bleomycin instillation was performed to construct the pulmonary fibrosis model after the immunization of Tocilizumab mimotopes. Lung histological analysis showed that the Tocilizumab mimotopes could significantly reduce the maladaptive repairment and abnormal remodeling. Immunoblotting assay and fluorescence staining showed that Immunization with the Tocilizumab mimotopes reduces the accumulation of fibrosis-related proteins. High level of lipid peroxidation product was observed in the animal model, while the Tocilizumab mimotopes vaccination could reduce the generation of lipid peroxidation product. Mechanism analysis further showed that Nrf-2 signaling, but not GPX-4 and FSP-1 signaling, was upregulated, and reduced the lipid peroxidation. Our results revealed that in the BLM-induced pulmonary fibrosis, high level of lipid peroxidation product was significantly accumulation in the lung tissues, which might lead to the occurrence of ferroptosis. The IL-6 pathway block therapy could inhibit lipid peroxidation product generation in the lung tissues by upregulating the Nrf-2 signaling, and further alleviate the pulmonary fibrosis.

Keywords: IL-6; Pulmonary fibrosis; Tocilizumab; fibroblast; lipid peroxidation; mimotope.

Figure 1.

Tocilizumab mimotopes alleviated bleomycin induced pulmonary fibrosis (a) Procedure of model construction and mimotope vaccination. (b) The tissue sections were thawed, dried, and then fixed with 4% paraformaldehyde PBS. The sections were deparaffinized by immersion in xylene, followed by dehydration in ethanol. Fibrosis was evaluated using Masson trichrome and H&E staining (×200 magnification, presented bar: 50 μm). The results showed that vaccination of tocilizumab mimotope could significantly reduce the pulmonary fibrosis. (mean ± SEM; ***p < .001; **p < .005; *p < .05; n.s. non-significant; Representative image, n = 6 mice per group).

Figure 2.

Immunization with the Tocilizumab mimotopes reduced the accumulation of fibrosis-related proteins (a) The tissue sections were thawed, dried, and then fixed with 4% paraformaldehyde PBS. Paraffin-embedded sections were deparaffinized by immersion in xylene, followed by dehydration in ethanol. After antigen retrieval, the sections were incubated with the primary antibodies. To detect primary antibodies, Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary antibody were added. Representative images were captured with a microscope (×200 magnification, presented bar: 50 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (collagen I): black (15.0) gamma (0.8) white (130.0); red (α-SMA): black (5.0) gamma (1.0) white (30.0); red (TGF-β): black (5.0) gamma (0.8) white (100). (b) Tissue protein extraction was performed with tissue extraction reagent I. Lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% bovine albumin and then treated with the primary and secondary antibodies. The membranes were scanned using a chemiluminescence/fluorescence image analysis system. The results showed that Tocilizumab mimotope immunization could significantly reduce the expressions of TGF-β, α-SMA and collagen I. (mean ± SEM; ***p < .001; **p < .005; *p < .05; n.s. non-significant; Representative image, n = 6 mice per group).

Figure 3.

Immunization with the Tocilizumab mimotopes reduced the proportion of lung fibroblasts the tissue sections were incubated with anti-sca-1 antibody and followed by Alexa Fluor 594-conjugated secondary antibody. Representative images were captured with a microscope (×200 magnification, presented bar: 50 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (SCA-1): black (15.0) gamma (1.0) white (70.0). The results showed that immunization with the Tocilizumab mimotopes could reduce the proportion of fibroblasts in BLM-induced model. (mean ± SEM; **p < .005; n.s. non-significant; Representative image, n = 6 mice per group).

Figure 4.

The Tocilizumab mimotopes reduced lipid peroxidation product in the whole lung tissues the tissue sections were incubated with anti-sca-1 and anti-MDA antibody and followed by Alexa Fluor 488 and 594-conjugated secondary antibody. Representative images were captured with a microscope (×200–400 magnification, presented bar: 50–20 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (SCA-1): black (10.0) gamma (0.6) white (80.0). Green (MDA): black (25.0) gamma (0.2) white (200.0). The results showed that immunization with the Tocilizumab mimotopes could reduce the expression of MDA in the whole lung tissue, including the fibroblasts. (Representative image, n = 6 mice per group. Mean ± SEM; ***p < .001; **p < .005; *p < .05; n.s. non-significant; Representative image, n = 6 mice per group).

Figure 5.

The Tocilizumab mimotopes did not increase the expression of GPX-4 in the whole lung tissues the tissue sections were incubated with anti-sca-1 and anti-GPX-4 antibody and followed by Alexa Fluor 488 and 594-conjugated secondary antibody. Representative images were captured with a microscope (×200–400 magnification, presented bar: 50–20 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (SCA-1): black (10.0) gamma (0.7) white (100.0). Green (GPX-4): black (30.0) gamma (0.1) white (100.0). The results showed that immunization with the Tocilizumab mimotopes could not increase the expression of GPX-4 in the whole lung tissues, including the fibroblasts in BLM-induced model. (Representative image, n = 6 mice per group. Mean ± SEM; ***p < .001; **p < .005; *p < .05; n.S. non-significant; Representative image, n = 6 mice per group).

Figure 6.

The Tocilizumab mimotopes did not increase the expression of FSP-1 in the whole lung tissues the tissue sections were incubated with anti-sca-1 and anti-FSP-1 antibody and followed by Alexa Fluor 488 and 594-conjugated secondary antibody. Representative images were captured with a microscope (×200–400 magnification, presented bar: 50–20 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (SCA-1): black (40.0) gamma (0.1) white (65.0). Green (FSP-1): black (25.0) gamma (0.5) white (65.0). The results showed that immunization with the Tocilizumab mimotopes did not increase the expression of FSP-1 in the whole lung tissues, including the fibroblasts in BLM-induced model. (Representative image, n = 6 mice per group. Mean ± SEM; ***p < .001; **p < .005; *p < .05; n.s. non-significant; Representative image, n = 6 mice per group).

Figure 7.

The Tocilizumab mimotopes increased the expression of nrf-2 whole lung tissues the tissue sections were incubated with anti-sca-1 and anti-nrf-2 antibody and followed by Alexa Fluor 488 and 594-conjugated secondary antibody. Representative images were captured with a microscope (×200–400 magnification, presented bar: 50–20 μm). Each image of immunofluorescence was acquired at the same adjustment parameters. Red (SCA-1): black (5.0) gamma (0.1) white (40.0). Green (nrf-2): black (5.0) gamma (0.5) white (150.0). The results showed that immunization with the Tocilizumab mimotopes increase the expression of nrf-2 in the whole lung tissues. The increased nrf-2 could be observed in the fibroblasts (Representative image, n = 6 mice per group. Mean ± SEM; ***p < .001; **p < .005; *p < .05; n.S. non-significant; Representative image, n = 6 mice per group).

Figure 8.

Tocilizumab mimotopes induced the significant changes on MDA and nrf-2 in the lung tissues tissue protein extraction was performed with tissue extraction reagent I. Lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% bovine albumin and then treated with the primary and secondary antibodies. The membranes were scanned using a chemiluminescence/fluorescence image analysis system. The results showed that tocilizumab mimotope immunization could significantly induce the significant changes on MDA and nrf-2 in the lung tissues. (mean ± SEM; ***p < .001; **p < .005; *p < .05; n.s. non-significant; Representative image, n = 6 mice per group).