title: Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?
abstract: During intervertebral disc (IVD) injury and degeneration, annulus fibrosus (AF) cells experience large mechanical strains in a pro-inflammatory milieu. We hypothesized that TNF-α, an initiator of IVD inflammation, modifies AF cell mechanobiology via cytoskeletal changes, and interacts with mechanical strain to enhance pro-inflammatory cytokine production. Human AF cells (N=5, Thompson grades 2-4) were stretched uniaxially on collagen-I coated chambers to 0%, 5% (physiological) or 15% (pathologic) strains at 0.5Hz for 24h under hypoxic conditions with or without TNF-α (10ng/mL). AF cells were treated with anti-TNF-α and anti-IL-6. ELISA assessed IL-1β, IL-6, and IL-8 production and immunocytochemistry measured F-actin, vinculin and α-tubulin in AF cells. TNF-α significantly increased AF cell pro-inflammatory cytokine production compared to basal conditions (IL-1β:2.0±1.4-84.0±77.3, IL-6:10.6±9.9-280.9±214.1, IL-8:23.9±26.0-5125.1±4170.8pg/ml for basal and TNF-α treatment, respectively) as expected, but mechanical strain did not. Pathologic strain in combination with TNF-α increased IL-1β, and IL-8 but not IL-6 production of AF cells. TNF-α treatment altered F-actin and α-tubulin in AF cells, suggestive of altered cytoskeletal stiffness. Anti-TNF-α (infliximab) significantly inhibited pro-inflammatory cytokine production while anti-IL-6 (atlizumab) did not. In conclusion, TNF-α altered AF cell mechanobiology with cytoskeletal remodeling that potentially sensitized AF cells to mechanical strain and increased TNF-α-induced pro-inflammatory cytokine production. Results suggest an interaction between TNF-α and mechanical strain and future mechanistic studies are required to validate these observations.
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States. Electronic address: morakotl@gmail.com.
authors.forename: Morakot
authors.initials: M
authors.lastname: Likhitpanichkul
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States. Electronic address: olivia.torre@icahn.mssm.edu.
authors.forename: Olivia M
authors.initials: OM
authors.lastname: Torre
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States. Electronic address: jadry.gruen@stonybrookmedicine.edu.
authors.forename: Jadry
authors.initials: J
authors.lastname: Gruen
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States; Department of Biomedical Engineering, The City College of New York, New York, NY, United States. Electronic address: benjamin.walter@mssm.edu.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Walter
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States. Electronic address: andrew.hecht@mountsinai.org.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Hecht
authors.affiliation: Leni and Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, Box 1188, New York, NY 10029, United States. Electronic address: james.iatridis@mssm.edu.
authors.forename: James C
authors.initials: JC
authors.lastname: Iatridis
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000070616:Annulus Fibrosus / Q000166:cytology*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D008875:Middle Aged; D013314:Stress, Mechanical
chemical_list: D000199:Actins; D016207:Cytokines
keywords: Annulus fibrosus; Infliximab; Intervertebral disc degeneration; Mechanical stretch; Mechanotransduction; TNF-α