Mediterr J Rheumatol 2015; 26(1): 74-76
Upregulation of Dickkopf-1 skin expression following B cell depletion therapy associates with enhanced resolution of skin fibrosis in patients with systemic sclerosis

Introduction: Experimental and clinical data indicate that B cells may actively participate in the fibrotic process in the context of systemic sclerosis (SSc).  Recently, the Wnt pathway has emerged as a crucial regulator of fibrosis.  In scleroderma skin, the Wnt pathway is highly activated; this is linked to the fact that Dickkopf-1 (Dkk-1), an inhibitor of the Wnt pathway, is virtually absent from scleroderma skin.

Objective: To assess the effect of B cell depletion therapy on Dkk-1 skin expression.

Methods: Skin biopsies were obtained from 11 patients with SSc, prior to and 6 months following RTX administration (biopsies from 8 patients were used for immunohistochemical assessment and biopsies from 3 patients for gene expression analysis).  Skin biopsies were also obtained from 3 additional patients with SSc at baseline and at 6 months for control purposes (2 patients receiving cyclophosphamide and 1 no treatment).  Most of these patients were enrolled in a clinical study assessing the efficacy of RTX in SSc.  All biopsies were taken from lesional skin of the forearm.  Skin involvement was assessed both clinically by applying the MRSS tool and histologically.  Collagen accumulation was assessed by Masson’s thrichrome and computerized image analysis was used to quantify the results. Dkk-1 skin expression was assessed by immunohistochemistry and by RT-PCR.  Circulating Dkk-1 levels were measured using a solid phase immunoassay.

Results: In all baseline biopsies Dkk-1 had no expression in spindle like cells in the dermis.  However, following RTX administration, 4 patients exhibited a significant upregulation of Dkk-1 expression in spindle like cells. In the control biopsies Dkk-1 was undetectable in spindle like cells at 6 months.  TGFβ skin expression in the upper dermis was significantly attenuated following treatment (p=0.018).  However, the dowregulation of TGFβ skin expression was significantly more pronounced in the subgroup of patients (n=4) which showed Dkk-1 upregulation.  In this subgroup TGFβ was downregulated by a mean percentage of 50.88%  in sharp contrast to only 15.98%  in patients that did not show Dkk-1 upregulation (p=0.022).  In the subgroup of patients which exhibited upregulation of Dkk-1 skin expression, an enhanced response, in terms of resolution of skin fibrosis, was found. In these patients, histologic analysis revealed that Dkk-1 upregulation associated with a significant decrease in collagen accumulation in the upper dermis by a mean±SEM 49.47±10.63% compared to 18.18±6.67% in patients which did not exhibit Dkk-1 upregulation (p=0.04).  Histologic data matched the clinical data; MRSS at 1 year decreased by a median of 63.23% in the subgroup that exhibited Dkk-1 upregulation compared to only 28.08% in the subgroup with undetectable Dkk-1.  PFT’s improved following RTX treatment, irrespective of Dkk-1 skin expression.  Circulating levels of Dkk-1 did not change following RTX treatment (mean±SEM of OD: 0.17±0.04 vs 0.19±0.03 for patients with SSc and healthy subjects, respectively, p=ns).  Gene expression analysis in fibroblasts revealed that DKK1 was significantly upregulated following RTX treatment (p=0.03, Figure 5)

Conclusions: Upregulation of Dkk-1 skin expression associates with an enhanced resolution of skin fibrosis following B cell depletion therapy.  RTX may mediate its beneficial effects on fibrosis by increasing Dkk-1 skin expression potentially by a TGFβ-dependent mechanism.  This could potentially downregulate the Wnt pathway, a well known driver of fibrosis.