Hydrogen improves the immune system of lung cancer patientsScientific Research

original title: Two weeks of hydrogen inhalation can significantly reverse adaptive and innate immune system senescence patients with advanced non-small cell lung cancer: a self-controlled study.

Published on: 25/12/2020

Abstract

After standard therapy, traditional models of enhancing antitumor immunity include immune reconstitution (eg, adoptive immune cell therapy or immune-enhancing drugs) to prevent relapse. For patients with advanced NSCLC, we report here two targets, immunosenescence in advanced NSCLC and hydrogen inhalation for immune reconstitution. From July 1 to September 25, 2019, 20 patients with non-small cell lung cancer were enrolled to measure immunosenescence in peripheral blood lymphocyte subsets, including T cells, natural killer/natural killer T cells, and gamma delta-T Cells. A two-week hydrogen inhalation was performed while awaiting treatment-related tests. All patients inhaled a mixture of hydrogen (66.7%) and oxygen (33.3%) daily for 4 hours at a gas flow rate of 3 L/min.None of the patients received any standard treatment during hydrogen inhalation. After the preconditioning experiment, the main indicators of immunosenescence were observed. Abnormally high indices included depleted cytotoxic T cells, senescent cytotoxic T cells, and killer Vδ1 cells. After 2 weeks of hydrogen therapy, the number of exhausted and senescent cytotoxic T cells decreased to the normal range and the number of killer Vδ1 cells increased. Abnormally low indicators included functional helper and cytotoxic T cells, Th1, all natural killer T cells, natural killer and Vδ2 cells. After 2 weeks of hydrogen therapy, all six cell subsets increased within normal ranges. Current data suggest that immunosenescence in advanced non-small cell lung cancer affects nearly all lymphocyte subsets, and that 2 weeks of hydrogen treatment can significantly improve most of these metrics.The study was approved by the Ethics Committee of Fuda Cancer Hospital, Jinan University, China on December 7, 2018 (approval number Fuda20181207), and was registered with ClinicalTrials.gov on January 24, 2019 (ID: NCT03818347).

Original Publication

Introduction

The recurrence and metastasis of malignant tumors are difficult to control, and the main reasons can be attributed to three: 1) immune aging; 2) tumor-related factors; 3) cancer treatment itself. [1] Immunosenescence is characterized by the gradual degeneration of the immune system during natural aging. Prolonged tumor-bearing status also leads to depletion and senescence of cytotoxic lymphocytes, including T cells, natural killer (NK) cells, NKT cells, and γδ (γδ) T cells. [2], [3] are tumor factors associated with excessive tumor burden and severe suppression of immune function, including increased CD4+CD25+ regulatory T cells. [4] Currently, various conventional treatments have been shown to accelerate the recurrence and metastasis of residual tumors, including surgery, [5] radiotherapy, [6] chemotherapy, [7] and even general anesthesia. [8]

Lung cancer is associated with the highest global incidence of all cancers, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of cases. [9] Due to the lack of early symptoms of lung cancer, many patients are diagnosed at an advanced stage, and survival is usually 1 year. [10] Studies have shown that nearly 27% of patients undergoing surgery and chemotherapy die from recurrence and metastasis. [11] Therefore, before and after standard therapy, immune reconstitution is a necessary approach to prolong survival in patients with advanced cancer. [12].Adoptive immune cell replenishment, particular or general immunological activators, cancer vaccinations, and exercise are the main techniques for immune rebuilding now being used. [13] Hydrogen can permeate into lymphocyte mitochondria as an antioxidant gas and specifically scavenge oxygen free radicals. [14],[15] Additionally, it has been demonstrated that hydrogen inhalation decreases the expression of PD-1, a sign of cell exhaustion, on the surface of cytotoxic T lymphocytes in patients with advanced colorectal cancer, extending both progression-free survival and overall survival. [17] In this study, immunosenescence in advanced NSCLC patients was examined, and immunosenescence was reversed while the patients were waiting for therapy using hydrogen inhalation.

Subjects and Methods

This self-controlled study included 20 advanced NSCLC patients admitted at Fuda Cancer Hospital between July and September 2019 who had to wait two weeks for therapy. The following inclusion criteria were listed in the clinical trial that was registered (ClinicalTrials.gov, ID: NCT03818347; Registration Date: January 24, 2019): stage III or IV NSCLC diagnosed by imaging and pathology[18]; tumor number 1-6; maximum tumor length 2 cm; Karnofsky performance status (KPS) score 7019; expected survival time > 6 months; platelet count 80 109/L; white blood cell count 3 Patients having a cardiac pacemaker, brain metastases, grade 3 hypertension, diabetic complications, and severe cardiac and pulmonary dysfunction were excluded from the study. During the duration of hydrogen inhalation, none of the patients received any conventional care.

Ethical Statement

In order to guarantee that any concerns regarding the accuracy or integrity of any component of the work are duly investigated and addressed, the writers are responsible for all aspects of the work. On December 7, 2018, the Fuda Cancer Hospital’s Ethics Committee granted their permission for the study protocol (approval number Fuda20181207). Each participant gave their written, informed consent in accordance with the Declaration of Helsinki.

Hydrogen inhalation method

Hydrogen was delivered by a hydrogen-oxygen nebulizer (AMS-H-03, Shanghai Asclepius Meditec Co., Ltd., Shanghai, China). The patients remained situated or prostrate, and breathed in a blend of hydrogen (66.7%) and oxygen (33.3%) by means of a nasal tube or veil with a gas stream rate of 3 L/min. The hydrogen inward breath was proceeded for 4 hours per day for 2 weeks in a specialized treatment facility.

Pulmonary symptoms and KPS score

Before and after hydrogen therapy, the respiratory function was assessed by very experienced respiratory physicians using a pulmonary function tester (Autospiro AS-507; Minato Medical Science, Tokyo, Japan), pulmonary tumor-related symptoms and the KPS score were evaluated by resident doctors in charge.

Immunophenotype evaluation

Roughly 5 mL of fringe blood was extricated from elbow of all selected patients some time recently hydrogen inward breath, as well as at one and two weeks after hydrogen inward breath. Fringe blood monocyte cells were separated employing a Ficoll arrangement and labeled with fluorescent antibodies (BD Biosciences, San Jose, CA, USA). T lymphocytes, NK, NKT, and γδ T cells were analyzed utilizing stream cytometry (FACSanto II; BD Biosciences, San Jose, CA, USA) by a proficient third-party assessment center (Shuangzhi Purui Therapeutic Research facility Co., Ltd., Wuhan, Hubei Area, China).

Statistical analysis

The calculation strategy of test estimate is based on MedSci Test Measure instruments (MSST) computer program (MedSci, Shanghai, China). Pneumonic indications some time recently and after hydrogen inward breath were compared utilizing combined t-test. Respiratory work, KPS score, and immunoassays were compared by rehashed measures investigation of change and Bonferroni’s different comparison test. Measurable contrasts were demonstrated by P < 0.05. All examinations and figures were delivered utilizing GraphPad Crystal 5.0 (GraphPad computer program, San Diego, CA, USA).

RESULTS

Clinical data of NSCLC patients A total of 20 consecutive patients admitted to the hospital participated in this study. The clinicopathological data are listed in [Table 1].

Changes in the KPS score and pulmonary symptoms of NSCLC patients
All patients received hydrogen inhalation therapy for 2 weeks and experienced no treatment-related symptoms or side effects. The results of respiratory function, KPS score, and pulmonary symptoms at 2 weeks before and after hydrogen therapy are shown in the table [Table 1]. 2] as shown in the figure. At the end of hydrogen treatment, there were fewer people with different pulmonary symptoms and a statistically lower number of moderate coughs and mild shortness of breath.

Changes in T cell subsets of NSCLC patients undergoing hydrogen therapy

This part of the test consists of three components: CD4+ T cells, CD8+ T cells, and cytokine-secreting helper T (Th) cells. Six major markers of immunosenescence were identified prior to starting hydrogen therapy. Among these indices, abnormally low indices included percentages of functional helper and cytotoxic T cells, Th1, and follicular Th cells; abnormally high indices were associated with percentages of depleted and aged cytotoxic T cells [Figure 1] ]. In the CD4+ subgroup, functional Th cells returned to the normal range after 1 week of hydrogen inhalation, and increased significantly after 2 weeks (P<0.05).After 2 weeks of hydrogen therapy, both depleted Th cells and regulatory T cells were gradually decreased (both P<0.05) [Fig. 1]A. In the CD8+ subgroup, functional cytotoxic T cells returned to the normal range after 2 weeks of hydrogen inhalation (P<0.05); after 2 weeks of treatment, both exhausted and senescent cytotoxic T cells gradually returned to the normal range (both P<0.05). 0.05) [Figure 1]B. Among the cytokine-secreting Th subsets, only Th1 cells gradually increased close to the normal range after 2 weeks of hydrogen inhalation treatment (P < 0.05; [Fig. 1] C. There were no significant changes in other cells within 2 weeks after hydrogen inhalation treatment.

Figure 1: Immunoassays of T cell subsets before and after hydrogen inhalation in patients with non-small cell lung cancer. NOTE: (A) Test results for CD4+ subsets. (B) Test results of CD8+ subsets. (C) Test results of cytokine-secreting CD4+ subsets. The red parallel long line in the figure represents the normal range, the black short line represents the average value at each time point, and the pink cell name represents the abnormal index before hydrogen treatment. Bonferroni’s multiple comparison test analysis. *P<0.05. CXCR5: C-X-C chemokine receptor type 5; Th: helper T; Tfh: follicular helper T.

Changes in NKT and NK cells of NSCLC patients undergoing hydrogen therapy

The percentages of total NKT cells, activated NK, and killer NK subsets were below the normal range before starting hydrogen therapy [Figure 2]. After one week of hydrogen inhalation, the activated NK cell subset increased to the normal range [Figure 2]C. After two weeks, both total NKT and killer NK subsets were higher than pretreatment percentages (both P<0.05; [Fig. 2] A and [Fig. 2] D, and activated NK cells were significantly increased (P < 0.01; [Fig. 2]). ] ] C. The total number of NK cells did not change significantly within 2 weeks after hydrogen inhalation [Fig. 2] B.

Figure 2: Immunoassay of natural killer (NK)T and NK cells before and after hydrogen inhalation in non-small cell lung cancer patients. Note: (A) Changes in the number of NKT cells. (B–D) Test results of the NK subsets. The parallel red long lines in the figure represent the normal range, the black short lines represent the average value at each time point, and the pink cell names represent the abnormal indicators before hydrogen treatment. Data were analyzed by repeated measures analysis of variance followed by Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01.

Changes in γδ T cell subsets of NSCLC patients undergoing hydrogen therapy

Before starting hydrogen therapy, the percentages of Vδ1 cells and killer Vδ1 cells were above the normal range, and the percentage of Vδ2 cells was below the normal range [Figure 3]. After 1 week of hydrogen inhalation, the percentage of Vδ2 cells in γδT cells increased to the normal range [Fig. 3]C. After two weeks, the percentages of total γδT cells and Vδ2 cells were higher than those before treatment (both P<0.05; [Fig. 3]A and [Fig. 3]C, depleted Vδ2 cells were lower than those before treatment (P < 0.05; [Fig. 3]C; Depleted Vδ1 cells significantly decreased (P<0.001; [Fig. 3]B and Killer Vδ2 cells were significantly increased (P<0.01; [Fig. 3]C) other cells were not significantly increased under hydrogen for 2 weeks). change) inhalation.

Figure 3: Immunoassay of the gamma delta (γδ) T cell subsets before and after hydrogen inhalation in non-small cell lung cancer patients.
Note: (A) Change in the number of γδ T cells. (B) Test results of the Vδ1 subsets. (C) Test results of the Vδ2 subsets. The parallel red long lines in the figure represent the normal range, the black short lines represent the average value at each time point, and the pink cell names represent the abnormal indicators before hydrogen treatment. Data were analyzed by repeated measures analysis of variance followed by Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. NK: Natural killer ; PD-1: programmed cell death protein 1.

1.	Manjili MH. Tumor dormancy and relapse: from a natural byproduct of evolution to a disease state. Cancer Res. 2017;77:2564-2569.
2.	Pawelec G. Immunosenescence and cancer. Biogerontology. 2017;18:717-721.
3.	Tarazona R, Sanchez-Correa B, Casas-Avilés I, et al. Immunosenescence: limitations of natural killer cell-based cancer immunotherapy. Cancer Immunol Immunother. 2017;66:233-245.
4.	Singer K, Cheng WC, Kreutz M, Ho PC, Siska PJ. Immunometabolism in cancer at a glance. Dis Model Mech. 2018;11:dmm034272.
5.	Tohme S, Simmons RL, Tsung A. Surgery for cancer: a trigger for metastases. Cancer Res. 2017;77:1548-1552.
6.	Chen X, Zhang L, Jiang Y, et al. Radiotherapy-induced cell death activates paracrine HMGB1-TLR2 signaling and accelerates pancreatic carcinoma metastasis. J Exp Clin Cancer Res. 2018;37:77.
7.	Ramani VC, Vlodavsky I, Ng M, et al. Chemotherapy induces expression and release of heparanase leading to changes associated with an aggressive tumor phenotype. Matrix Biol. 2016;55:22-34.
8.	Sekandarzad MW, van Zundert AAJ, Lirk PB, Doornebal CW, Hollmann MW. Perioperative Anesthesia Care and Tumor Progression. Anesth Analg. 2017;124:1697-1708.
9.	Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69:7-34.
10.	Expert Panel on Thoracic Imaging, de Groot PM, Chung JH, et al. ACR Appropriateness Criteria® noninvasive clinical staging of primary lung cancer. J Am Coll Radiol. 2019;16:S184-S195.
11.	Yan X, Jiao SC, Zhang GQ, Guan Y, Wang JL. Tumor-associated immune factors are associated with recurrence and metastasis in non-small cell lung cancer. Cancer Gene Ther. 2017;24:57-63.
12.	Finn OJ. Immuno-oncology: understanding the function and dysfunction of the immune system in cancer. Ann Oncol. 2012;23 Suppl 8:viii6-viii9.
13.	Moore SC, Lee IM, Weiderpass E, et al. Association of leisure-time physical activity with risk of 26 types of cancer in 1.44 million adults. JAMA Intern Med. 2016;176:816-825.
14.	Kelso GF, Porteous CM, Coulter CV, et al. Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties. J Biol Chem. 2001;276:4588-4596.
15.	Ferrer MD, Sureda A, Tauler P, Palacín C, Tur JA, Pons A. Impaired lymphocyte mitochondrial antioxidant defences in variegate porphyria are accompanied by more inducible reactive oxygen species production and DNA damage. Br J Haematol. 2010;149:759-767.
16.	Kamphorst AO, Wieland A, Nasti T, et al. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017;355:1423-1427.
17.	Akagi J, Baba H. Hydrogen gas restores exhausted CD8+ T cells in patients with advanced colorectal cancer to improve prognosis. Oncol Rep. 2019;41:301-311.
18.	Osmani L, Askin F, Gabrielson E, Li QK. Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): Moving from targeted therapy to immunotherapy. Semin Cancer Biol. 2018;52:103-109.
19.	Thuluvath PJ, Thuluvath AJ, Savva Y. Karnofsky performance status before and after liver transplantation predicts graft and patient survival. J Hepatol. 2018;69:818-825.
20.	Klebanoff CA, Gattinoni L, Restifo NP. CD8+ T-cell memory in tumor immunology and immunotherapy. Immunol Rev. 2006;211:214-224.
21.	Klebanoff CA, Gattinoni L, Torabi-Parizi P, et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci U S A. 2005;102:9571-9576.
22.	Chen IH, Lai YL, Wu CL, et al. Immune impairment in patients with terminal cancers: influence of cancer treatments and cytomegalovirus infection. Cancer Immunol Immunother. 2010;59:323-334.
23.	Saavedra D, García B, Lorenzo-Luaces P, et al. Biomarkers related to immunosenescence: relationships with therapy and survival in lung cancer patients. Cancer Immunol Immunother. 2016;65:37-45.
24.	Owonikoko TK, Ragin CC, Belani CP, et al. Lung cancer in elderly patients: an analysis of the surveillance, epidemiology, and end results database. J Clin Oncol. 2007;25:5570-5577.
25.	Franceschi C, Valensin S, Fagnoni F, Barbi C, Bonafè M. Biomarkers of immunosenescence within an evolutionary perspective: the challenge of heterogeneity and the role of antigenic load. Exp Gerontol. 1999;34:911-921.
26.	Lanna A, Henson SM, Escors D, Akbar AN. The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells. Nat Immunol. 2014;15:965-972.
27.	Henson SM, Lanna A, Riddell NE, et al. p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8+ T cells. J Clin Invest. 2014;124:4004-4016.
28.	Akbar AN, Henson SM, Lanna A. Senescence of T lymphocytes: implications for enhancing human immunity. Trends Immunol. 2016;37:866-876.
29.	Garg AD, Agostinis P. Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses. Immunol Rev. 2017;280:126-148.
30.	Fulop T, Kotb R, Fortin CF, Pawelec G, de Angelis F, Larbi A. Potential role of immunosenescence in cancer development. Ann N Y Acad Sci. 2010;1197:158-165.
31.	Yang Y, Li B, Liu C, et al. Hydrogen-rich saline protects immunocytes from radiation-induced apoptosis. Med Sci Monit. 2012;18:Br144-148.
32.	Yang Y, Gao F, Zhang H, et al. Molecular hydrogen protects human lymphocyte AHH-1 cells against 12C6+ heavy ion radiation. Int J Radiat Biol. 2013;89:1003-1008.
33.	De La Cruz LM, Nocera NF, Czerniecki BJ. Restoring anti-oncodriver Th1 responses with dendritic cell vaccines in HER2/neu-positive breast cancer: progress and potential. Immunotherapy. 2016;8:1219-1232.
34.	Sun C, Sun HY, Xiao WH, Zhang C, Tian ZG. Natural killer cell dysfunction in hepatocellular carcinoma and NK cell-based immunotherapy. Acta Pharmacol Sin. 2015;36:1191-1199.
35.	Taniguchi M, Harada M, Dashtsoodol N, Kojo S. Discovery of NKT cells and development of NKT cell-targeted anti-tumor immunotherapy. Proc Jpn Acad Ser B Phys Biol Sci. 2015;91:292-304.
36.	Mao TL, Miao CH, Liao YJ, Chen YJ, Yeh CY, Liu CL. Ex Vivo Expanded Human Vγ9Vδ2 T-Cells Can Suppress Epithelial Ovarian Cancer Cell Growth. Int J Mol Sci. 2019;20:1139.
37.	Davey MS, Willcox CR, Joyce SP, et al. Clonal selection in the human Vδ1 T cell repertoire indicates γδ TCR-dependent adaptive immune surveillance. Nat Commun. 2017;8:14760.
38.	Fisher JPH, Yan M, Heuijerjans J, et al. Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells. Clin Cancer Res. 2014;20:5720-5732.
39.	Ohue Y, Nishikawa H. Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target? Cancer Sci. 2019;110:2080-2089.