Physical inactivity is associated with decreased growth differentiation factor 11 in chronic obstructive pulmonary disease

Rie Tanaka,1 Hisatoshi Sugiura,1 Mitsuhiro Yamada,1 Tomohiro Ichikawa,1 Akira Koarai,1 Naoya Fujino,1 Satoru Yanagisawa,1 Katsuhiro Onodera,1 Tadahisa Numakura,1 Kei Sato,1 Yorihiko Kyogoku,1 Hirohito Sano,1 Shun Yamanaka,1 Tatsuma Okazaki,1 Tsutomu Tamada,1 Motohiko Miura,2 Tsuneyuki Takahashi,3 Masakazu Ichinose1 1Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan; 2Department of Respiratory Medicine, Tohoku Rosai Hospital, Aoba-ku, Sendai, Japan; 3Department of Internal Medicine, Tohoku Medical and Pharmaceutical University Wakabayashi Hospital, Wakabayashi-ku, Sendai, Japan Background: Growth differentiation factor 11 (GDF11) is reported to possess anti-aging and rejuvenating effects, including muscle regeneration and to be highly expressed in skeletal muscle. Recently, we demonstrated that the levels of plasma GDF11 were decreased in COPD. However, the effect of decreased circulating GDF11 in the pathophysiology of COPD remains unknown. The aim of this study is to investigate the association between the plasma GDF11 levels and various clinical parameters in patients with COPD. Patients and methods: Eighteen ex-smokers as control subjects and 70 COPD patients participated in the current study. We measured the levels of plasma GDF11 using immunoblotting, lung function, physical activity using a triaxial accelerometer, quadriceps strength, exercise capacity, and systemic inflammatory markers. We investigated the association between the levels of plasma GDF11 and these clinical parameters. Results: The levels of plasma GDF11 in the COPD patients had significant positive correlations with the data of lung function. Furthermore, the levels of plasma GDF11 were significantly correlated with the physical activity, quadriceps strength, and exercise capacity. Moreover, the levels of plasma GDF11 were significantly correlated with the data of inflammatory markers. Although various factors were related to GDF11, the multiple regression analysis showed that physical activity was significantly associated with the levels of plasma GDF11. Conclusion: Physical inactivity was significantly related to the decreased GDF11 levels in COPD, which might be useful for understanding the pathogenesis of COPD. Clarifying the relationships between the physical inactivity and GDF11 may reveal a potentially attractive therapeutic approach in COPD via increasing the plasma levels of GDF11. Keywords: physical activity, muscle strength, rejuvenating factor, COP

FIGURE 2 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb induces degranulation of iNKT cells toward K562 cells. PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. Sorted iNKT cells were maintained in the presence of IL2 overnight and then washed. iNKT cells were then maintained in the presence of IL2 until analysis. A, Sorted iNKT cells were maintained overnight and then the culture supernatant was collected. The culture supernatant depleted with anti-mouse Ig beads or the control was added to iNKT cell cultures overnight prior to coculture with K562 cells for the CD107a assay. B, Sorted iNKT cells treated with the 6B11 mAb (10, 50, and 100 ng/ mL) or isotype mAb (mIgG1, 50 ng/mL) were cocultured with K562 cells for CD107a assays. C, Sorted iNKT cells treated with the 6B11 mAb (50 ng/mL) or isotype mAb (50 ng/mL) were cocultured with K562 cells at E/T ratios of 10:1, 5:1, and 2.5:1. An LDH release assay was then performed. D, Sorted iNKT cells treated with the 6B11 mAb (50 ng/ mL) or isotype (mIgG1, 50 ng/mL) were cocultured with K562 cells. Culture supernatants were collected for Cytometric Bead Array to assess IFNγ and TNFα production. E, iNKT cells were treated with the 6B11 mAb and anti-CD3e (UCHT1), anti-CD28, anti-CD2, or isotype control Abs overnight. iNKT cells were then washed and cocultured with K562 cells. Data are representative of two independent experiments. Data represent the mean ± SEM. *, P P P P < 0.0001. HD, healthy donor.</p

FIGURE 3 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb treatment induces degranulation of iNKT cells toward some cell lines. A, The indicated cell lines were stained with an anti-CD1d mAb and analyzed by flow cytometry. B, PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. iNKT cells sorted by MACS were maintained in the presence of IL2 overnight, and then iNKT cells were washed twice and cultured in fresh medium with IL2 for 4–5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) or isotype (mIgG1) were cocultured with the indicated cell lines at an E/T ratio of 2:1. A CD107a assay was then performed. Data are representative of two independent experiments.</p

FIGURE 5 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

NK cell–activating receptors and costimulatory molecules are involved in degranulation of 6B11-mAb bound iNKT cells toward K562 cells. A and B, iNKT cells sorted using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, washed twice, and then cultured in fresh medium with IL2 for 4 or 5 days. A, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cross-linked with goat anti-mouse IgG (10 µg/ mL) or cocultured with K562 cells (control). A CD107a assay was then performed. B, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with K562 cells at an E/T ratio of 2:1 in the presence of anti-NKG2D, anti-DNAM1, anti-2B4, anti-LFA-1, anti-LFA-2, or isotype (mIgG, 10 µg/mL) Abs. A CD107a assay was then performed. Data are representative of two independent experiments. Data represent the mean ± SEM. ****, P < 0.0001. HD, healthy donor.</p

FIGURE 6 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

Primary tumor cells express CD32. A, Expression levels of FCGR2A across TCGA tumors were assessed by GEPIA. TCGA study abbreviations: LAML, acute myeloid leukemia; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; KIRP, kidney renal papillary carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; STAD, stomach adenocarcinoma; UCS, uterine carcinosarcoma. B, Lung tumor tissue arrays were subjected to IHC to detect CD32 expression in LUAD and LUSC (×400). C, LAML cells were stained with anti-CD32 or isotype control Abs and analyzed by flow cytometry. D, iNKT cells sorted using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, washed twice, and then cultured in fresh medium with IL2 for 4 or 5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with LAML or K562 cells (control) at an E/T ratio of 2:1. A CD107a assay was then performed. PT, patient.</p

FIGURE 7 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

CD32-expressing normal cells barely induce degranulation of 6B11 mAb- bound NKT cells. A, PBMCs isolated from healthy donors were stained with Abs against FC receptors (CD16, CD32, and CD64), a monocyte marker (CD14), B-cell marker (CD19), and neutrophil marker (CD66b). Stained cells were analyzed by flow cytometry. Data are representative of three independent experiments. B, iNKT cells sorted using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, washed twice, and then cultured in fresh medium with IL2 for 4 or 5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with monocytes, B cells, neutrophils, or K562 cells (control) at an E/T ratio of 2:1. A CD107a assay was then performed. HD, healthy donor.</p

TABLE 1 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

IHC scoring of CD32 in the patients with lung cancer</p