Unraveling the signaling pathways promoting fibrosis PNAS PLUS in Dupuytren’s disease reveals TNF as a therapeutic target Liaquat S. Verjeea, Jennifer S. N. Verhoekxa,b, James K. K. Chana, Thomas Krausgrubera, Vicky Nicolaidoua, David Izadia, Dominique Davidsonc, Marc Feldmanna,1, Kim S. Midwooda, and Jagdeep Nanchahala,1 aKennedy Institute of Rheumatology, University of Oxford, London W6 8LH, United Kingdom; bDepartment of Plastic and Reconstructive Surgery, Erasmus Medical Centre, 3015, Rotterdam, The Netherlands; and cDepartment of Plastic Surgery, St John’s Hospital, Livingstone EH54 6PP, United Kingdom Contributed by Marc Feldmann, January 18, 2013 (sent for review December 5, 2012) Dupuytren’sdiseaseisaverycommonprogressivefibrosis of the characteristically express α-smooth muscle actin (α-SMA), which palm leading to flexion deformities of the digits that impair hand is the actin isoform typical of vascular smooth muscle cells (6). function. The cell responsible for development of the disease is the Fibroblast to myofibroblast differentiation is characterized by myofibroblast. There is currently no treatment for early disease or for α-SMA expression, and exposure of the cells to stress leads to the preventing recurrence following surgical excision of affected tissue incorporation of α-SMA protein into stress fibers (7). Unlike in in advanced disease. Therefore, we sought to unravel the signaling granulation tissue, where the expression of α-SMA is transient (8), pathways leading to the development of myofibroblasts in Dupuyt- α-SMA expression is persistent in Dupuytren’s disease. The de- ren’s disease. We characterized the cells present in Dupuytren’s tis- velopment of myofibroblasts has been shown to be dependent on sue and found significant numbers of immune cells, including a number of different environmental cues, including tension in the fl classically activated macrophages. High levels of proin ammatory matrix and exposure to a variety of different soluble mediators (7). ’ cytokines were also detected in tissue from Dupuytren s patients. The best studied of these is TGF-β1. The expression of TGF-β1 We compared the effects of these cytokines on contraction and and associated signaling molecules is elevated in Dupuytren’s fi fi pro brotic signaling pathways in broblasts from the palmar and disease (9), and Dupuytren’s myofibroblasts or dermal fibroblasts nonpalmar dermis of Dupuytren’s patients and palmar fibroblasts MEDICAL SCIENCES ’ from the same patients proliferated in response to 1 or 5 ng/mL from non-Dupuytren s patients. Exogenous addition of TNF, but not TGF-β1 (10). Normal human dermal fibroblasts cultured in other cytokines, including IL-6 and IL-1β, promoted differentiation stressed collagen lattices also showed increased α-SMA expres- into specifically of palmar dermal fibroblasts from Dupuytren’s sion when treated with 1 ng/mL TGF-β1 (11). Fibroblasts derived patients in to myofibroblasts. We also demonstrated that TNF acts fi from the transverse carpal ligament of patients unaffected by via the Wnt signaling pathway to drive contraction and pro brotic ’ β signaling in these cells. Finally, we examined the effects of targeted Dupuytren s disease exposed to 2 ng/mL TGF- 1 showed no increase in α-SMA staining (12). However, these fibroblasts, as cytokine inhibition. Neutralizing antibodies to TNF inhibited the ’ fi contractile activity of myofibroblasts derived from Dupuytren’s well as Dupuytren s myo broblasts, did exhibit increased iso- patients, reduced their expression of α-smooth muscle actin, and metric contraction of collagen lattices when exposed to 12.5 ng/ β mediated disassembly of the contractile apparatus. Therefore, we mL of TGF- 1, with higher doses being inhibitory (13). There- β α showed that localized inflammation in Dupuytren’s disease contrib- fore, TGF- 1 can promote cell proliferation and increase -SMA utes to the development and progression of this fibroproliferative expression and matrix contraction in a manner dependent on disorder and identified TNF as a therapeutic target to down-regu- both the dose and the tissue of origin of the cells. late myofibroblast differentiation and activity. Inflammation is also known to play a crucial role in fibrosis, and various proinflammatory cytokines have been implicated in driving musculoskeletal | scarring Significance upuytren’s disease is a common fibroproliferative disorder Dwith a prevalence >7% in the United States (1). The classic Fibrosis, a hallmark of many clinical disorders, occurs because of description of disease progression is the initial appearance of uncontrolled myofibroblast activity. We studied Dupuytren’s palmar nodules characterized by high cellularity and cell pro- disease, a common hereditable fibrotic condition that causes the liferation, followed by the development of cords. This phase is fingers to irreversibly curl toward the palm. We found that followed by a final fibrotic stage that is associated with matura- freshly isolated tissue from Dupuytren’s patients contained tion of the cords and digital contractures resulting in significant macrophages and released proinflammatory protein mediators impairment of hand function (2). Established flexion deformities (cytokines). Of the cytokines, only TNF selectively converted of the digits are most commonly treated by surgical excision normal fibroblasts from the palm of patients with Dupuytren’s (fasciectomy) of the cord. The long recovery time following disease into myofibroblasts via activation of the Wnt signaling surgery has led to description of alternative techniques of dis- pathway. Conversely, blockade of TNF resulted in reversal of the rupting the cord with a needle (percutaneous fasciotomy) (3) or myofibroblast phenotype. Therefore, TNF inhibition may pre- enzymatic digestion using collagenase injections (4). However, vent progression or recurrence of Dupuytren’s disease. even following surgery, patients often have significant residual dysfunction due to irreversible fixed flexion deformities of the Author contributions: M.F., K.S.M., and J.N. designed research; L.S.V., J.S.N.V., J.K.K.C., T.K., fi V.N., and D.I. performed research; D.D. and J.N. collected human tissue samples; L.S.V., J.S.N.V., joints. Currently, there is no speci c treatment for early disease J.K.K.C., T.K., V.N., D.I., D.D., M.F., K.S.M., and J.N. analyzed data; and L.S.V., J.S.N.V., J.K.K.C., or prevention of recurrence following fasciectomy or fasciotomy T.K., V.N., D.I., D.D., M.F., K.S.M., and J.N. wrote the paper. ’ of Dupuytren s cords. Therefore, we sought to unravel the cel- The authors declare no conflict of interest. lular mechanisms leading to the development of this disease to 1To whom correspondence may be addressed. E-mail: [email protected] reveal novel potential therapeutic targets. or [email protected]. The cell responsible for matrix deposition and contraction This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in Dupuytren’s disease is the myofibroblast (5). Myofibroblasts 1073/pnas.1301100110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301100110 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 the progression of fibrotic diseases (14). Histological studies have Results identified the presence of immune cells in Dupuytren’s disease; in Cells in Dupuytren’s Nodules and the Cytokines They Produce. To particular, the number of macrophages was shown to correlate quantify the presence of immune cells in Dupuytren’s tissue, flow fi with the quantity of myo broblasts (15). Intralesional steroid cytometric analysis was performed on cells directly disaggregated fl injections led to temporary softening and attening of Dupuyt- from Dupuytren’s nodules. The majority (87 ± 6%) of cells were ’ ren s nodules (16) and reduced recurrence following percutaneous myofibroblasts, together with a significant number of macro- needle fasciotomy (17). It has been suggested that this therapeutic phages (6.5 ± 2.3%), with the classically activated (CD68+/ fi ’ bene t of steroids in early Dupuytren s disease may be due to CD163−) M1 phenotype predominating (4.8 ± 2.2%; Fig. 1 A and diminished leukocyte recruitment (18), as well as increased apo- B). Immunohistochemistry showed that the macrophages were ptosis of macrophages and fibroblasts, with reduced proliferation distributed throughout the Dupuytren’s nodules (Fig. 1C) and of the latter (19). However, the link between disease progression clustered around the blood vessels. Serial histological sections and localized inflammation in Dupuytren’s tissue remains unclear, revealed no neutrophil elastase positive cells. Soluble cytokines and molecular mechanisms by which inflammatory cytokines di- fi produced by freshly disaggregated Dupuytren’s nodule tissue in- rectly drive myo broblast differentiation remain unknown. β ± ± To explore these issues, we systematically studied the cytokines cluded variable amounts of TGF- 1 (mean SD: 236 248 pg/ – ± ± produced by freshly disaggregated cells from Dupuytren’s nod- mL; range, 4 852 pg/mL), as well as TNF (mean SD: 78 26 pg/ ± ± ules. TNF was identified as a key regulator of the myofibroblast mL), IL-6 (mean SD: 5,591 3,215 pg/mL), and consistent with phenotype, and TNF blockade in vitro led to down-regulation of the presence of substantial numbers of classically activated M1 the myofibroblast phenotype. Our data indicate that progression macrophages, GM-CSF (mean ± SD: 64 ± 24 pg/mL). Low levels of of early palmar nodules of Dupuytren’s disease to deposition of IL-1β (mean ± SD: 17 ± 10 pg/mL), IL-10 (mean ± SD: 11 ± 9pg/ extracellular matrix in cords and subsequent digital contractures mL), and IFNγ (mean ± SD: 7 ± 6 pg/mL) were observed (Fig. 1D). in vivo may be prevented by local administration of a TNF in- Cells from the same patient samples were also maintained in hibitor. This approach may also be effective in preventing re- culture and examined for constituent cells and cytokine profiles currence following surgery, percutaneous needle fasciotomy, or at passage 2. Macrophages were absent, and the passaged cells collagenase treatment.