Online Supplement for the Manuscript: Supplementary Material and Methods
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Supplementary material Ann Rheum Dis Online Supplement for the manuscript: The acyltransferase skinny hedgehog regulates TGF-dependent fibroblast activation in SSc by Ruifang Liang1, Rosebeth Kagwiria1, Ariella Zehender1, Clara Dees1, Christina Bergmann1, Andreas Ramming1, Dorota Krasowska2, Małgorzata Michalska-Jakubus2, Alexander Kreuter3, Georg Schett1, Jörg H. W. Distler1 Supplementary Material and Methods Patient and Public Involvement Patients were not involved in the design of the study or the interpretation of the results, but donated biopsies for this study. The results of the study will be presented on congress to enable distribution also by patients. Patients and fibroblasts Dermal fibroblasts were isolated from skin biopsies of 23 SSc patients and 21 matched healthy volunteers. Biopsies were taken at the forearm, 15 ± 2 cm away from the styloid processus. All patients fulfilled the 2013 ACR/EULAR criteria for SSc.[1] Sixteen patients were female, seven were male. The median age of SSc patients was 45 years (range: 19-65 years), and their median disease duration was 5 years (range: 0.5-10 years). All patients and healthy volunteers signed a consent form approved by the local institutional review board. Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis Murine models of SSc Three different murine models of SSc were employed: Bleomycin-induced, TBRact-induced and Topoisomerase 1 (topo)-induced dermal fibrosis. Bleomycin was injected every other day at concentrations of 0.5 mg/ml as described [2, 3]. Injections with the vehicle, 0.9% NaCl served as controls. For TBRact-induced fibrosis, 4-week-old mice received of 6.67 × 107 pfu/mouse of replication-deficient type 5 adenoviruses encoding for constitutively active TBRI construct into defined areas of at the upper back four times every other week [4]. Injections of 6.67 × 107 pfu/mouse of replication-deficient type 5 adenoviruses encoding for LacZ served as controls. In the Topo model, fibrosis was induced by four local injections of topo every other week. Recombinant human topo was diluted to 500 units/ml and mixed with Complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, Taufkirchen, Germany) 3:2 (volume/volume). 250µl were injected subcutaneously into C57BL/6 mice (female, 6 weeks old) in defined areas of the upper back [5]. Injections of the vehicle, including CFA served as controls. Reporter assay NIH3T3-Light2 cells were kindly provided by Prof. Dr. Suzanne Eaton (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Cells were transfected with siRNA against Hhat or non-targeting control siRNA (n.t.siRNA) by nucleofection as described [6]. 24h after transfection, cells were serum starved and stimulated with recombinant TGF. The fluorescence intensity was measured at a Luminoskan Ascent Microplate-Luminometer (ThermoFisher, Bonn, Germany). Coculture systems In transwell co-culture assays, direct cell-cell contact was prevented by using cell culture insert with a 0.4 µm pore size polycarbonate membrane (Falcon) in a 24-well plate. 3 x 104 of Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis SHH-light II reporter cells were cultured in the bottom of the well (100% confluence), whereas 3 x 104 NIH3T3 fibroblasts transfected with n.t.siRNA or Hhat siRNA were placed in the insert of 24-well plate (100% confluence). NIH3T3 cells were serum starved for 24 h and infected with adenoviruses encoding for LacZ or TBR. 24 hours after infection, the insert was removed, SHH-light II reporter cells were lysed and subsequent analyzed by Dual- Luciferase assays (Supplementary Fig. 3 A). In direct co-culture system, 2 x 104 of NIH3T3 cells transfected with Hhat siRNA or n.t. siRNA and 2 x 104 of SHH-light II (together with the NIH3T3 cells 100% confluency) cells were placed in a single well of 24-well plate. Cells were serum starved for 24 h and infected with adenoviruses LacZ or TBR. 24 h after infection, cells were lysed and Dual-luciferase were performed (Supplementary Fig. 3B). siRNA-mediated knockdown of HHAT and Smad3 Complexes of siRNA and atelocollagen (Koken, Tokyo, Japan) were prepared as described previously [7, 8]. The following siRNA duplexes (Thermo Fisher) were used: mHhat: 5´- GUUAAGAGAAGGUGGUACAUU-3´, antisense 5´-PUGUACCACCUUCUCUUAACUU- 3´. Non-targeting siRNA duplexes served as controls. Atelocollagen/siRNA complexes were injected intracutaneously once weekly. Analysis of dermal fibrosis and adverse effects Dermal thickness, -smooth muscle actin and hydroxyproline content were analyzed as described previously [2, 3, 9-14]. Mice were monitored daily for activity, weight loss, texture of the fur and diarrhea. Quantitative real time-PCR Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis Gene expression was quantified by SYBR-Green real-time-PCR using the MX3005P Detection System (Agilent Technologies, Santa Clara CA, US).[15, 16] Samples without enzyme in the reverse transcription reaction, without template and dissociation curve analysis served as controls. All primers are summarized in supplementary table 1. Western blotting Protein samples were separated by SDS-polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking, membranes were incubated with polyclonal antibodies against HHAT (Santa Cruz, Heidelberg, Germany) overnight at 4°C. For the detection of SHH, polyclonal antibodies against SHH (Santa Cruz) were used. Membranes were incubated with horseradish- peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark). Conditioned cell supernatants from fibroblasts transfected with Hhat siRNA or non-targeting siRNA were collected and centrifuged at 500 g for 15 minutes to remove cellular debris. The supernatants were concentrated by using Amicon Ultra-0.5 mL Centrifugal Filters 50 kDa to enrich for oligomeric SHH (75 kDa, 120 kDa, 180 kDa) [17]. The flow-through was concentrated by trichloroacetic acid precipitation to enrich for monomeric SHH (19 kDa). After addition of reducing SDS sample buffer, samples were heated at 95 °C for 10 minutes, briefly centrifuged at 14,000 x g for 5 minutes, and loaded onto the 12% (for oligomeric SHH) and 6% (for monomeric SHH) SDS-PAGE. We confirmed these findings by an additional approach. In this approach, proteins in the supernatants were cross-linked with 1% PFA for 10min. The supernatants were then applied to Amicon Ultra-0.5 mL Centrifugal Filters and the flow-through was precipitated by TCA and further preceded as described above. Immunofluorescence staining Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis Paraffin-embedded skin sections or cultured fibroblasts were stained with antibodies against prolyl-4-hydroxylase- (P4H), -smooth muscle actin (SMA) (Sigma-Aldrich), HHAT (Sigma-Aldrich), GLI2 and DAPI. Myofibroblasts were identified as single, spindle shaped cells in the dermis positive for -smooth muscle actin. Concentration-matched species- specific immunoglobulins (Vector Laboratories) served as control antibodies. The staining was analyzed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Statistics All in vitro data are presented as median with interquartile range (IQR), and all in vivo data as dot blots. Differences between the groups were tested by non-parametric Mann-Whitney U test. P-values less than 0.05 were considered as significant. Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis Supplementary Figures Supplementary Figure 1: Hedgehog signaling is active in fibroblasts expressing HHAT. A, Co-stainings of HHAT with the hedgehog transcription factor GLI2 and the fibroblast marker prolyl-4-hydroxylase- (P4H) at 200-fold magnification. Semi-quantitative analysis of HHAT staining in fibroblasts in the skin of SSc patients and healthy volunteers (n=5 for SSc patients and 5 for matched healthy controls). B, immunofluorescence staining (400-fold and 1000-fold magnification) with quantification in healthy and SSc patients skins by two independent human observers (n=6). Supplementary Figure 2: TGF induces HHAT expression in a SMAD3-dependent manner. A-B, Effects of siRNA-mediated knockdown of SMAD3 on the mRNA (A) and protein (B) levels of HHAT in human dermal fibroblasts (n=4 biological replicates with ≥2 technical replicates).C-D, Costaining for Hhat, Smad3, Vimentin and DAPI in murine models of skin fibrosis with quantification (C) and representative images (D) (200-fold and 600-fold magnification n=5). Supplementary Figure 3: Knockdown of Hhat abrogates TBR-induced long-range hedgehog signaling. A, Transwell assays with physical separation of fibroblasts and reporter cells and activation of the reporter exclusively by long-range hedgehog signaling; schematic presentation of the experiment (left) and quantification of the reporter activity (right). B,Direct coculture of reporter cells and fibroblasts. Reporter activity is mediated by short- and long-range hedgehog Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273. doi: 10.1136/annrheumdis-2019-215066 Supplementary material Ann Rheum Dis signaling. Schematic presentation of the experiment (left) and quantification of the reporter activity (right). Fibroblasts were transfected with a constitutively active TGF receptor type I (TBR) (or LacZ) and Hhat siRNA (or scrambled siRNA) (n=10). C, Western blot analysis of oligomeric and monomeric forms of SHH in the supernatant of fibroblasts transfected with a constitutively active TGF receptor type I (TBR) (or LacZ) and Hhat siRNA (or scrambled siRNA) in direct coculture and transwell settings. One representative WB (coculture) and quantification (n=4 independent experiments for each setting). Supplementary Figure 4: Knockdown efficacy of Hhat in murine skin. Protein levels of Hhat in the skin of bleomycin- (A) or Topo-challenged (B) mice with or without atellocollagen/siRNA complexes (n=6). Liang R, et al. Ann Rheum Dis 2019; 0:1269–1273.