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.
Introduction
Tocilizumab is a recombinant humanized monoclonal antibody which binds to soluble and membrane- IL-6 receptor (IL-6 R) and blocks the downstream signaling of IL-6.Citation1,Citation2 Tocilizumab is now licensed for the treatment of rheumatoid arthritis (RA),Citation3 systemic juvenile idiopathic arthritis (sJIA), polyarticular juvenile idiopathic arthritis (pJIA),Citation4,Citation5 Castleman’s diseaseCitation6,Citation7 and represents an inspiring efficacy in clinic applications. Meanwhile, Tocilizumab is on the road to be an alternative treatment of other inflammatory conditions such as Crohn’s disease,Citation8 systemic lupus erythematosus,Citation9 Takayasu arteritis (TA),Citation10 giant cell arteritis (GCA),Citation11 polymyalgia rheumatica (PMR),Citation12 refractory adult-onset Still disease,Citation13 and early systemic sclerosis-associated interstitial lung disease.Citation14 In cancer, IL-6 signaling activated by tumor-associated macrophages contributes to tumor growth and development by modulating immunosuppression and angiogenesis.Citation15–17 As a result, the administration of Tocilizumab is demonstrated effective in the treatment of cancer. Since 2020, coronavirus disease (COVID-19) pandemic extends the Tocilizumab application to control the secondary cytokine release syndrome (CRS) and severe COVID-19.Citation18,Citation19
However, therapeutic antibodies still have practical limitations. One important limitation is that these biologic drugs are expensive. Persist blocking activity needs repeatedly administration. Thus, patient access to these monoclonal antibodies has always been excluded because of the high cost. Cost effectiveness is still the prime consideration of patient when choosing a biologic therapy. Another important limitation is patient compliance, IV infusion are generally administered on hospital infusion units, which means that patients need accept routine walk to hospital persistently. Although subcutaneous injection is a more favorable option, not all patients are eligible because of the inadequate training and support.Citation20,Citation21
Mimotope, a kind of peptide vaccine, has been developed to overcome these limitations. Due to immune tolerance, the use of a natural epitope peptide alone is inefficient at inducing an immune reaction.Citation22 Mimotope structurally mimic the given antibody-binding site of the natural epitope peptide but are composed of different amino acids.Citation23,Citation24 Active immunization induced by the mimotope can initiate the ongoing production of the desired antibodies, which recognize not only the mimicked epitope but the natural receptor.
We have demonstrated that the mimotope of Tocilizumab could alleviate the renal fibrosis by interfering with both IL-6 and ferroptosis signaling.Citation25 However, the effect of the vaccination of Tocilizumab mimotopes on the pulmonary fibrosis was not investigated. Thus, we sought to observe the changes triggered by the Tocilizumab mimotopes vaccination in the Bleomycin-induced animal model.
Materials and methods
Mimotope preparation and immunization
The Tocilizumab mimotope (KTMSAEEFDNWLGGGSGGGS)Citation26 was synthesized chemically with a purity of > 98%. A segment peptide (AIPLVVPFYSHSGGGSGGGS) in the M13 phage from the phage display library (E8110S, NEB) served as a control. Mice were immunized through subcutaneous injection with 100 μg of the Tocilizumab mimotope and control peptide on days −52, −31, and −10. Complete Freund adjuvant/incomplete Freund adjuvant (CFA/IFA) (F5881/F5506, Merck) was used as the adjuvant in all groups.
Construction of animal model
Six-week-old male mice (C57BL/6J) from Vital River Laboratory (Beijing, China) were bred in the specific pathogen-free (SPF) facility at the Animal Science Center of Baotou Medical College. Ethical approval for use of animals in research was obtained from the Ethics Committee of the First Affiliated Hospital of Baotou Medical College. All procedures involving animals were performed according to the Research Animal Administration Guidelines of China and the Guidelines for the Care and Use of Laboratory Animals in China.
Briefly, mice were anesthetized by intravenous injection of pentobarbital sodium (35 mg/kg) and then intratracheally administrated with Bleomycin (BLM, 5 mg/kg, dissolved in 100 μl of PBS) on day 0. Mice were divided into either the PBS group, BLM group, BLM + control peptide immunization group (BLM+CP), or BLM + Tocilizumab mimotope immunization group (BLM+TM). The procedure of peptide immunization and model construction was shown in . Mice were sacrificed on day 28 after BLM instillation, and lung tissues were harvested for subsequent experiments.
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).
Lung histological analysis
The tissue sections were thawed, dried, and then fixed with 4% paraformaldehyde PBS for 30 min. Paraffin-embedded sections were deparaffinized by immersion in xylene, followed by dehydration in ethanol. Pulmonary fibrosis was evaluated using Masson trichrome and hematoxylin and eosin (H&E) staining. The severity of fibrosis was graded histologically using the method proposed by Ashcroft T et al. All the lung sections were scored blindly and independently by at least two independent observers.Citation27 Representative images were captured with a microscope (DM3000, Leica, Germany).
Fluorescence staining
The tissue sections were thawed, dried, and then fixed with 4% paraformaldehyde PBS for 30 min. Paraffin-embedded sections were deparaffinized by immersion in xylene, followed by dehydration in ethanol. After antigen retrieval, the sections were incubated with anti-α-SMA (19245, Cell Signaling Technology), -Collagen I (260043, Abcam), -nuclear factor erythroid 2-related factor 2 (Nrf-2) (80593–1-RR, proteintech), -Malondialdehyde (MDA) (243066, Abcam), -Glutathione Peroxidase 4 (GPX-4) (125066, Abcam), -Stem Cell Antigen-1 (SCA-1) (109211, Abcam), -TGF-β (215715, Abcam) and Ferroptosis Suppressor Protein-1 (FSP-1) (sc -377,120, Santa Cruz Biotechnology). To detect primary antibodies, Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary antibody (A-11005, A-11008, ThermoFisher) were added for 30 min. Representative images were captured with a microscope (DM3000, Leica, Germany).
Immunoblotting analysis
Tissue protein extraction was performed with Tissue Extraction Reagent I (Invitrogen). Tissues lysates mixed with sodium dodecyl sulfate (SDS) sample buffer 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 anti-α-SMA (19245, Cell Signaling Technology), -collagen I (260043, Abcam), -TGF-β (215715, Abcam), -MDA (243066, Abcam), -GPX-4 (59735, Cell Signaling Technology), and -Nrf-2 (16396–1-AP, proteintech). The primary antibodies were detected with a horseradish peroxidase (HRP)-conjugated secondary antibody (7074/7076, CST) diluted 1:3000 for 1 h at room temperature. The membranes were scanned using a chemiluminescence/fluorescence image analysis system (Tanon, Shanghai, China).
Statistical analyses
Image Pro Plus 6.0 software (Media Cybernetics) and ImageJ software (version 1.53e; NIH) were used for histology analysis. The positive value was determined by measuring the integrated optical density (IOD) or Integrated Density of staining regions in every image. For the immunoblotting assay, ImageJ software (version 1.53e; NIH) was used to quantify the intensity of the bands. GraphPad Prism 8.0 (GraphPad Software) software was used for statistical analyses. The data are expressed as the mean ± standard error of mean (SEM). Differences between groups were evaluated for statistical significance using one-way analysis of variance (ANOVA) or t test, and statistical significance was set at p < .05.
Results
The tocilizumab mimotopes alleviated pulmonary fibrosis in BLM-induced model
We first evaluated the pulmonary fibrosis of mice from each group with an analysis of Masson trichrome staining. As shown in , a significant increase collagen accumulation could be observed in the lungs at day 28. As we expected, additional vaccination of Tocilizumab mimotope could significantly reduce the collagen deposition induced by BLM instillation. In staining showed that the thickness of alveolar wall and fibrotic scarring emerged in the lung of BLM treated mice, while the formation of these morphological structure disorders decreased by Tocilizumab mimotope.
The Tocilizumab mimotopes reduced the accumulation of fibrosis-related proteins BLM-induced model
Fibrosis can be characterized through the detections of TGF-β, α-SMA and collagen I. Thus, we detected the expressions of fibronectin, α-SMA and collagen I in the lung tissues by Immunoblotting and Fluorescence staining. As shown in , we found Tocilizumab mimotope immunization could significantly reduce the expressions of TGF-β, α-SMA and collagen I at day 28 BLM instillation.
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).
The tocilizumab mimotopes decreased fibroblasts BLM-induced model
Since the aim of this study is to evaluate the changes on fibroblasts in animal model under the administration of the Tocilizumab mimotopes, we next performed the fluorescence staining to observe the proportion of fibroblasts. Sca-1, an immunophenotypically distinct mouse lung fibroblast marker, was used to recognize the fibroblasts.Citation28 The results showed that immunization with the Tocilizumab mimotopes could reduce the proportion of fibroblasts in BLM-induced model ().
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).
The Tocilizumab mimotopes reduced the level of lipid peroxidation in the whole lung tissues
Products of lipid peroxidation play the vital role in ferroptosis. Thus, we detected the MDA, an important product of lipid peroxidation. The results showed that the level of MDA significantly increased not only in the fibroblasts, but also in the whole lung tissues, while additional vaccination of the Tocilizumab mimotopes could reduce the high level of MDA ().
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).
The Tocilizumab mimotopes did not increase the expression of GPX-4 and FSP-1 in the whole lung tissues
GPX4 and FSP-1 pathways are major antioxidant mechanisms to inhibit lipid peroxidation. We detected the levels of GPX-4 and FSP-1 to further confirm the lipid peroxidation changes induced by the Tocilizumab mimotopes vaccination. The results showed that The Tocilizumab mimotopes did not increase the levels of GPX-4 and FSP-1 in the whole lung tissues ().
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).
The Tocilizumab mimotopes increased the expression of nrf-2 in the whole lung tissues
Nrf-2 is another antioxidant signaling, which can also play a part in preventing ferroptosis. We consequently detected the expression of Nrf-2 after negative changes on GPX-4 and FSP-1. In this event, the significant increased expression of Nrf-2 was observed in the whole lung tissues from the mice with the Tocilizumab mimotopes immunization. Additionally, the increased Nrf-2 could be observed in the fibroblasts ().
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).
The Tocilizumab mimotopes changes the expression of MDA and nrf-2 in the lung tissues
Immunoblotting assay was performed to observe the expression of MDA, GPX-4, FSP-1 and Nrf-2 in the whole lung tissues. The results were similar to the fluorescence staining, the vaccination of the Tocilizumab mimotopes could reduce the high level of MDA and increase the expression of Nrf-2 in the lung tissues ().
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).
Discussion
Interleukin-6 (IL-6), a multifunctional cytokine, maintains the balances of immune regulation, hematopoiesis, inflammation and oncogenesis.Citation29 IL-6 secreted from macrophages and its’ soluble receptor are elevated in mouse models of lung fibrosis,Citation30 and blockade of IL-6 can attenuate the pulmonary fibrosis in animal model.Citation31–37 In patients with pulmonary fibrosis, IL-6 is demonstrated to exert mitogenic effects on fibroblasts isolated from the patients.Citation38 Further mechanism analysis showed that the MAPKs, canonical TGF-β signaling, JAK/STAT3 signaling, and histone deacetylation may be the downstream signaling of IL-6 in idiopathic pulmonary fibrosis. Recently, novel studies seeking for the endotypes of pulmonary fibrosis are conducted in BLM induced animal model because the treatment with biologic agents of pulmonary fibrosis in future is based on patients’ endotype and by taking into consideration patients’ comorbidities.Citation39,Citation40 PD-1/PD-L1 axis is considered to be a potential therapeutic target for idiopathic pulmonary fibrosisCitation41 and anti-IL-6 regimen in patients with increased IL-6 also toward a precision medicine approach.Citation42
As expected, the Tocilizumab mimotopes vaccination could alleviate the pulmonary fibrosis in BLM-induced model. Then we investigated what changes would happen in the lung fibroblast induced by the vaccination. Fibroblasts are the most common cell type throughout the body and play crucial roles in the tissue remodeling process by producing an appropriate extracellular matrix (ECM) They are also the central mediators of the pathological fibrotic accumulation of ECM. Unlimited fibroblast proliferation and differentiation in response to prolonged tissue injury and chronic inflammation cause excessive repair of the tissue, which leads to the tissue fibrosis.Citation43,Citation44 The results showed that the Tocilizumab mimotope administration could significantly decrease the proportion of lung fibroblasts and reduce the pulmonary fibrosis. Then we observed the level of MDA, a major product of lipid peroxidation which plays a key role in ferroptosis. Increased accumulation of MDA leads to the changes of membrane thickness and permeabilization by inducing membrane pores formation and membrane barrier function destroy. Meanwhile, increased accumulation of MDA could further increase more hydrophilic acyl tails peroxidative lipids, which would bend to the water phase, causing membrane instability, micelle formation, and finally cell death.Citation45 Intriguedly, the vaccination of Tocilizumab mimotope seemed to inhibit, but not promote the lipids peroxidation in the whole lung tissues, including the unlimited fibroblasts. One might be that the mimotopes actually induced a higher level of MDA in the fibroblasts soon after BLM instillation so that the fibroblasts might undergo ferroptosis from the early stage of the model. On day 28 after BLM instillation, most of the fibroblasts might die and we hardly observe the fibroblasts with a high level of MDA in the lung tissues. Another possible explanation was that a high level of MDA might lead to the ferroptosis of fibroblasts, which further promoted pulmonary fibrosis. It is reported that the ferroptosis can help glioblastoma cells reveal aggressive behaviors and predict a poor survival.Citation46 We assumed fibroblasts in pulmonary fibrosis might have a similar behavior pattern to tumor cells that the ferroptosis of the fibroblasts might promote the fibrosis. The fibroblasts consequently released various cytokines to recruit and activate immune cells, aggravate inflammation, and create a profibrotic environment.
We then evaluated the levels of GPX-4, FSP-1, and Nrf-2, three important antioxidant pathways in lipids peroxidation. The GPX-4 and FSP-1 pathways are two major antioxidant systems. GPX-4 pathway catalyzes the reduction of peroxidized lipids either in free form or in complex into lipid alcohols.Citation47 FSP-1 pathway catalyzes the regeneration of ubiquinone (Coenzyme Q10, CoQ10), which directly scavenge lipid peroxyl radicals and reduce lipid hydroperoxides.Citation48,Citation49 Recently, Nrf-2 was reported to play a role in regulating several antioxidant genes, including glutathione regulation, NADPH regeneration, and iron regulation. Nrf2 activation protect cells against oxidative stress by indirectly modulates the lipids peroxidation.Citation50 The inhibition of IL-6 signaling has been correlated with the activation of Nrf-2 recently.Citation51–53 Our results showed that the Tocilizumab mimotopes inhibited the lipids peroxidation in the whole lung tissues independent of GPX4 and FSP-1 signaling, but by up-regulating Nrf-2. Because the presence of fibroblasts was significantly suppressed by the Tocilizumab mimotopes, the increased Nrf-2 was only observed in these sporadic fibroblasts. Further experiments would be performed to confirm the changes of Nrf-2 in the fibroblasts.
In conclusion, the lipids peroxidation may result in the fibroblasts ferroptosis, and further promote the pulmonary fibrosis in BLM-induced animal model. The immunization of the Tocilizumab mimotopes inhibit the lipids peroxidation in the lung tissues and alleviate the pulmonary fibrosis by upregulating the Nrf-2 signaling.
Author contribution statement
Jin Guo participated in the experiments and the manuscript preparation. Li Bai constructed the animal model. Haoming Song, Lin Yang and Li Bai conceived the study, participated in the analysis and interpretation of data.
Disclosure statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability statement
All data that support the findings of this study are available within the article upon reasonable request.
Additional information
Funding
References
- Sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig MM. Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth. Cancer Res. 1993 Feb 15;53(4):851–11.
- Tanaka T, Narazaki M, Kishimoto T. Therapeutic targeting of the interleukin-6 receptor. Annu Rev Pharmacol Toxicol. 2012;52:199–219. doi:10.1146/annurev-pharmtox-010611-134715
- Scott LJ. Tocilizumab: A Review in Rheumatoid Arthritis. Drugs. 2017 Nov;77(17):1865–79. doi:10.1007/s40265-017-0829-7.
- Yokota S, Imagawa T, Mori M, Miyamae T, Aihara Y, Takei S, Iwata N, Umebayashi H, Murata T, Miyoshi M. et al. Efficacy and safety of tocilizumab in patients with systemic-onset juvenile idiopathic arthritis: a randomised, double-blind, placebo-controlled, withdrawal phase III trial. Lancet. 2008 Mar 22;371(9617):998–1006. doi:10.1016/S0140-6736(08)60454-7.
- De Benedetti F, Brunner HI, Ruperto N, Kenwright A, Wright S, Calvo I, Cuttica R, Ravelli A, Schneider R, Woo P. et al. Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis. N Engl J Med. 2012 Dec 20;367(25):2385–95. doi:10.1056/NEJMoa1112802.
- Nishimoto N, Sasai M, Shima Y, Nakagawa M, Matsumoto T, Shirai T, Kishimoto T, Yoshizaki K. Improvement in Castleman’s disease by humanized anti-interleukin-6 receptor antibody therapy. Blood. 2000 Jan 1;95(1):56–61. doi:10.1182/blood.V95.1.56.
- Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, Nishioka K. et al. Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease. Blood. 2005 Oct 15;106(8):2627–32. doi:10.1182/blood-2004-12-4602.
- Ito H, Takazoe M, Fukuda Y, Hibi T, Kusugami K, Andoh A, Matsumoto T, Yamamura T, Azuma J, Nishimoto N. A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn’s disease. Gastroenterology. 2004 Apr;126(4):989–96. discussion 947. doi:10.1053/j.gastro.2004.01.012.
- Illei GG, Shirota Y, Yarboro CH, Daruwalla J, Tackey E, Takada K, Fleisher T, Balow JE, Lipsky PE. Tocilizumab in systemic lupus erythematosus: data on safety, preliminary efficacy, and impact on circulating plasma cells from an open-label phase I dosage-escalation study. Arthritis Rheum. 2010 Feb;62(2):542–52. doi:10.1002/art.27221. PMID: 20112381; PMCID: PMC3057537.
- Abisror N, Mekinian A, Lavigne C, Vandenhende MA, Soussan M, Fain O. Club Rhumatismes et Inflammation, and SNFMI. Tocilizumab in refractory Takayasu arteritis: a case series and updated literature review. Autoimmun Rev. 2013 Oct;12(12):1143–9. doi:10.1016/j.autrev.2013.06.019.
- Spiera R, Unizony SH, Bao M, Luder Y, Han J, Pavlov A, Stone JH. Tocilizumab vs placebo for the treatment of giant cell arteritis with polymyalgia rheumatica symptoms, cranial symptoms or both in a randomized trial. Semin Arthritis Rheum. 2021 Apr;51(2):469–476. doi:10.1016/j.semarthrit.2021.03.006.
- Devauchelle-Pensec V, Berthelot JM, Cornec D, Renaudineau Y, Marhadour T, Jousse-Joulin S, Querellou S, Garrigues F, De Bandt M, Gouillou M. et al. Efficacy of first-line tocilizumab therapy in early polymyalgia rheumatica: a prospective longitudinal study. Ann Rheum Dis. 2016 Aug;75(8):1506–10. doi:10.1136/annrheumdis-2015-208742.
- Kaneko Y, Kameda H, Ikeda K, Ishii T, Murakami K, Takamatsu H, Tanaka Y, Abe T, Takeuchi T. Tocilizumab in patients with adult-onset still’s disease refractory to glucocorticoid treatment: a randomised, double-blind, placebo-controlled phase III trial. Ann Rheum Dis. 2018 Dec;77(12):1720–1729. doi:10.1136/annrheumdis-2018-213920.
- Roofeh D, Lin CJF, Goldin J, Kim GH, Furst DE, Denton CP, Huang S, Khanna D; focuSsced investigators. Tocilizumab prevents progression of early systemic sclerosis-associated interstitial lung disease. Arthritis Rheumatol. 2021 Jul;73(7):1301–10. doi:10.1002/art.41668.
- Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004 Jan;4(1):71–8. doi:10.1038/nrc1256.
- Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009 Mar;9(3):162–74. doi:10.1038/nri2506.
- Santer FR, Malinowska K, Culig Z, Cavarretta IT. Interleukin-6 trans-signalling differentially regulates proliferation, migration, adhesion and maspin expression in human prostate cancer cells. Endocr Relat Cancer. 2010 Feb 18;17(1):241–53. doi:10.1677/ERC-09-0200.
- Coomes EA, Haghbayan H. Interleukin-6 in covid-19: a systematic review and meta-analysis. Rev Med Virol. 2020 Nov;30(6):1–9. doi:10.1002/rmv.2141.
- Karampitsakos T, Papaioannou O, Tsiri P, Katsaras M, Katsimpris A, Kalogeropoulos AP, Malakounidou E, Zarkadi E, Tsirikos G, Georgiopoulou V. et al. Tocilizumab versus baricitinib in hospitalized patients with severe COVID-19: an open label, randomized controlled trial. Clin Microbiol Infect. 2023 Mar;29(3):372–378. doi:10.1016/j.cmi.2022.10.015.
- Sheppard M, Laskou F, Stapleton PP, Hadavi S, Dasgupta B. Tocilizumab (Actemra). Hum Vaccin Immunother. 2017 Sep 2;13(9):1972–88. doi:10.1080/21645515.2017.1316909.
- Huynh TK, Ostergaard A, Egsmose C, Madsen OR. Preferences of patients and health professionals for route and frequency of administration of biologic agents in the treatment of rheumatoid arthritis. Patient Prefer Adherence. 2014 Jan 20;8:93–9. doi:10.2147/PPA.S55156.
- Adams TE. Tolerance to self-antigens in transgenic mice. Mol Biol Med. 1990;7:341–57.
- Felici F, Luzzago A, Folgori A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the bordetella pertussis toxin from phage peptide libraries. Gene. 1993 Jun 15;128(1):21–7. doi:10.1016/0378-1119(93)90148-v.
- Luzzago A, Felici F, Tramontano A, Pessi A, Cortese R. Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene. 1993 Jun 15;128(1):51–7. doi:10.1016/0378-1119(93)90152-s.
- Yang L, Guo J, Yu N, Liu Y, Song H, Niu J, Gu Y. Tocilizumab mimotope alleviates kidney injury and fibrosis by inhibiting IL-6 signaling and ferroptosis in UUO model. Life Sci. 2020 Nov 15;261:118487. doi:10.1016/j.lfs.2020.118487.
- Yang L, Xing R, Li C, Liu Y, Sun L, Liu X, Wang Y. Active immunization with tocilizumab mimotopes induces specific immune responses. BMC Biotechnol. 2015 Jun 2;15:46. doi:10.1186/s12896-015-0161-9.
- Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988 Apr;41(4):467–70. doi:10.1136/jcp.41.4.467.
- Long KK, Montano M, Pavlath GK. Sca-1 is negatively regulated by TGF-beta1 in myogenic cells. FASEB J. 2011 Apr;25(4):1156–65. doi:10.1096/fj.10-170308.
- Tanaka T, Narazaki M, Ogata A, Kishimoto T. A new era for the treatment of inflammatory autoimmune diseases by interleukin-6 blockade strategy. Semin Immunol. 2014 Feb;26(1):88–96. doi:10.1016/j.smim.2014.01.009.
- Hu L, Yu Y, Huang H, Fan H, Hu L, Yin C, Li K, Fulton DJ, Chen F. Epigenetic regulation of interleukin 6 by histone acetylation in macrophages and its role in paraquat-induced pulmonary fibrosis. Front Immunol. 2017 Jan 30;7:696. doi:10.3389/fimmu.2016.00696.
- Wang Y, Sang X, Shao R, Qin H, Chen X, Xue Z, Li L, Wang Y, Zhu Y, Chang Y. et al. Xuanfei Baidu decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway. J Ethnopharmacol. 2022 Jan 30;283:114701. doi:10.1016/j.jep.2021.114701.
- Epstein Shochet G, Brook E, Bardenstein-Wald B, Shitrit D. TGF-β pathway activation by idiopathic pulmonary fibrosis (IPF) fibroblast derived soluble factors is mediated by IL-6 trans-signaling. Respir Res. 2020 Feb 18;21(1):56. doi:10.1186/s12931-020-1319-0.
- Xue Z, Zhao F, Sang X, Qiao Y, Shao R, Wang Y, Gao S, Fan G, Zhu Y, Yang J. Combination therapy of tanshinone IIA and puerarin for pulmonary fibrosis via targeting IL6-JAK2-STAT3/STAT1 signaling pathways. Phytother Res. 2021 Oct;35(10):5883–5898. doi:10.1002/ptr.7253.
- Pulivendala G, Bale S, Godugu C. Honokiol: a polyphenol neolignan ameliorates pulmonary fibrosis by inhibiting TGF-β/Smad signaling, matrix proteins and IL-6/CD44/STAT3 axis both in vitro and in vivo. Toxicol Appl Pharmacol. 2020 Mar 15;391:114913. doi:10.1016/j.taap.2020.114913.
- Le TT, Karmouty-Quintana H, Melicoff E, Le TT, Weng T, Chen NY, Pedroza M, Zhou Y, Davies J, Philip K. et al. Blockade of IL-6 Trans signaling attenuates pulmonary fibrosis. J Immunol. 2014 Oct 1;193(7):3755–68. doi:10.4049/jimmunol.1302470. Epub 2014 Aug 29.
- El-Kashef DH, Youssef ME, Nasr M, Alrouji M, Alhajlah S, AlOmeir O, El Adle Khalaf N, Ghaffar DMA, Jamil L, Abdel-Nasser ZM. et al. Pimitespib, an HSP90 inhibitor, augments nifuroxazide-induced disruption in the IL-6/STAT3/HIF-1α autocrine loop in rats with bleomycin-challenged lungs: evolutionary perspective in managing pulmonary fibrosis. Biomed Pharmacother. 2022 Sep;153:113487. doi:10.1016/j.biopha.2022.113487.
- Han D, Gong H, Wei Y, Xu Y, Zhou X, Wang Z, Feng F. Hesperidin inhibits lung fibroblast senescence via IL-6/STAT3 signaling pathway to suppress pulmonary fibrosis. Phytomedicine. 2023 Apr;112:154680. doi:10.1016/j.phymed.2023.154680.
- Moodley YP, Scaffidi AK, Misso NL, Keerthisingam C, McAnulty RJ, Laurent GJ, Mutsaers SE, Thompson PJ, Knight DA. Fibroblasts isolated from normal lungs and those with idiopathic pulmonary fibrosis differ in interleukin-6/gp130-mediated cell signaling and proliferation. Am J Pathol. 2003 Jul;163(1):345–54. doi:10.1016/S0002-9440(10)63658-9.
- Tzouvelekis A, Antoniou K, Kreuter M, Evison M, Blum TG, Poletti V, Grigoriu B, Vancheri C, Spagnolo P, Karampitsakos T. et al. 2021 Jan 25. The DIAMORFOSIS (DIAgnosis and management of lung canceR and FibrOSIS) survey: international survey and call for consensus. ERJ Open Res. 7(1):00529–2020. doi:10.1183/23120541.00529-2020.
- Tzouvelekis A, Karampitsakos T, Kourtidou S, Bouros E, Tzilas V, Katsaras M, Antonou C, Dassiou M, Bouros D. Impact of depression on patients with idiopathic pulmonary fibrosis. Front Med. 2020 Feb 7;7:29. doi:10.3389/fmed.2020.00029.
- Karampitsakos T, Galaris A, Chrysikos S, Papaioannou O, Vamvakaris I, Barbayianni I, Kanellopoulou P, Grammenoudi S, Anagnostopoulos N, Stratakos G. et al. Expression of PD-1/PD-L1 axis in mediastinal lymph nodes and lung tissue of human and experimental lung fibrosis indicates a potential therapeutic target for idiopathic pulmonary fibrosis. Respir Res. 2023 Nov 14;24(1):279. doi:10.1186/s12931-023-02551-x.
- Karampitsakos T, Juan-Guardela BM, Tzouvelekis A, Herazo-Maya JD. Precision medicine advances in idiopathic pulmonary fibrosis. EBioMedicine. 2023 Sep;95:104766. doi:10.1016/j.ebiom.2023.104766.
- Phan SH. Biology of fibroblasts and myofibroblasts. Proc Am Thorac Soc. 2008 Apr 15;5(3):334–7. doi:10.1513/pats.200708-146DR.
- Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol. 2014 May 27;5:123. doi:10.3389/fphar.2014.00123.
- Wong-Ekkabut J, Xu Z, Triampo W, Tang IM, Tieleman DP, Monticelli L. Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study. Biophys J. 2007 Dec 15;93(12):4225–36. doi:10.1529/biophysj.107.112565.
- Yee PP, Wei Y, Kim SY, Lu T, Chih SY, Lawson C, Tang M, Liu Z, Anderson B, Thamburaj K. et al. Neutrophil-induced ferroptosis promotes tumor necrosis in glioblastoma progression. Nat Commun. 2020 Oct 27;11(1):5424. doi:10.1038/s41467-020-19193-y.
- Chen JJ, Galluzzi L. Fighting resilient cancers with iron. Trends Cell Biol. 2018 Feb;28(2):77–78. doi:10.1016/j.tcb.2017.11.007.
- Morré DJ, Morré DM. Non-mitochondrial coenzyme Q. Biofactors. 2011 Sep-Oct;37(5):355–60. doi:10.1002/biof.156.
- Ernster L, Forsmark-Andrée P. Ubiquinol: an endogenous antioxidant in aerobic organisms. Clin Investig. 1993;71(8 Suppl):S60–5. doi: 10.1007/BF00226842. PMID: 8241707.
- Lee C. Collaborative power of Nrf2 and PPARγ activators against metabolic and drug-induced oxidative injury. Oxid Med Cell Longev. 2017;2017:1378175. doi:10.1155/2017/1378175
- Lee J, Lim JW, Kim H. Lycopene inhibits IL-6 expression by upregulating NQO1 and HO-1 via activation of Nrf2 in ethanol/lipopolysaccharide-stimulated pancreatic acinar cells. Antioxid (Basel). 2022 Mar 8;11(3):519. doi:10.3390/antiox11030519.
- Luo JF, Zhou H, Lio CK. Akebia saponin D inhibits the inflammatory reaction by inhibiting the IL-6-STAT3-DNMT3b Axis and activating the Nrf2 pathway. Molecules. 2022 Sep 22;27(19):6236. doi:10.3390/molecules27196236.
- Narimiya T, Kanzaki H, Yamaguchi Y, Wada S, Katsumata Y, Tanaka K, Tomonari H. Nrf2 activation in osteoblasts suppresses osteoclastogenesis via inhibiting IL-6 expression. Bone Rep. 2019 Nov 1;11:100228. doi:10.1016/j.bonr.2019.100228.