Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

Cell culture and reagents. Primary cultures of fibroblasts were established by explanations from SSc and healthy adult skin, neonatal foreskin, and newborn mouse skin from WT and RP105-KO mice. Briefly, the skin biopsies were digested using 0.2% collagenase (Sigma-Aldrich, C0130), in DMEM with 20% FBS at 37°C for 3 hours, centrifuged, and washed; the skin tissue was resuspended with DMEM with 20% FBS. The skin tissue was transferred gently into 100 mm sterile-treated Petri dishes, followed by scraping off the epidermis using sterile scalpel. Next, the tissue was chopped into fine pieces, and explanted fibroblasts were allowed to grow in DMEM with 20% FBS. The media was replaced until the explanted fibroblasts were confluent. Low-passage fibroblasts were grown in monolayers in plastic dishes and studied at early confluence. Cultures were maintained in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco BRL), 1% vitamin solutions, and 2 mM glutamine. All other tissue culture reagents were from Lonza. For experiments, cultures were placed in serum-free media containing 0.1% bovine serum albumin overnight, followed by treatment with LPS (InvivoGen), TGF-β1 (Peprotech), or human Tenascin-C (Sigma-Aldrich) or transfection with pDUO-MD1/RP105 (InvivoGen) for up to 72 hours.

To generate bone marrow–derived macrophages and dendritic cells, femurs and tibias from WT (C57BL/6J) and RP105-KO mice were harvested and cultured as described previously (34, 35). To evaluate the expression of IL-6 on macrophages, dendritic cells, and whole blood, cells and whole blood were incubated with LPS at indicated concentrations for 24 hours. IL-6 expression from the culture supernatants and mice sera were assayed using an ELISA (R&D Systems, catalog M6000B).

Isolation and analysis of RNA. At the end of the experiments, total RNA was isolated and reverse transcribed to cDNA using Supermix (Quanta Biosciences, cDNA Synthesis Supermix) as described previously (18). The products (20 ng) were amplified using SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Prism Sequence Detection System. Data were normalized to GAPDH RNA, and fold change in samples was calculated. Sequences of the primers are shown in Table 2.

Cell migration assay. The effect of RP105 on fibroblast function was further evaluated by an in vitro wound-healing assay. Briefly, human skin fibroblasts from both WT and RP105-KO mice were seeded on serum-free DMEM, and confluent monolayers were mechanically wounded using p1000 pipette tips. Following incubation of the cultures with TGF-β for indicated periods, wound gap widths (μm) were determined at indicated time points at 6 randomly selected sites per high-powered field (hpf). The experiments were performed in 2 different skin fibroblasts lines.

Western analysis. At the end of the experiments, cultures were harvested and washed with PBS, followed by addition of RIPA lysis buffer mixed with phosphatase and protease inhibitors. Cells were lysed and spun down and were subjected to protein estimation bicinchoninic acid assay using bovine serum albumin as standard for comparison. Then, equal volumes of cell lysates (20 μg/lane) were denatured and subjected to Western analysis using primary antibodies specific for type I collagen (Southern Biotechnology) and tubulin (Sigma-Aldrich, T5168), followed by appropriate secondary antibodies as described previously (18). Membranes were then exposed to enhanced chemiluminescence detection using ECL Reagent (Pierce). Band intensities were quantitated using Image J software (NIH) and corrected for tubulin in each lane.

Transfection study. Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours. Cultures were harvested, and whole-cell lysates were assayed for Western analysis (6). The experiment was repeated twice with consistent results.

PLA. The PLA was carried out using Sigma Duolink reagents (Sigma-Aldrich, DUO92009). and fibroblasts from healthy controls and patients with SSc were cultured in 8-well chamber slides, fixed in ice-cold methanol for 5 minutes, and subjected to blocking using Duolink blocking solution (Sigma-Aldrich, DUO92009) for 1 hour at 37°C in a humidity chamber. For TLR4 homodimerization assay, anti-TLR4 (mouse, Invitrogen, PA5-23124, 1:200) and ant-TLR4 (rabbit, Santa Cruz, sc293072, 1:100) were used, while for testing RP105-TLR4 dimerization, anti TLR4 (mouse, Invitrogen, PA5-23124, 1:200) and anti-RP105 (rabbit, Abcam, ab184956, 1:100) were used. Slides were incubated overnight at 4°C in the primary antibodies diluted in Duolink antibody diluent (Sigma-Aldrich) followed by washing twice with (5 minutes) in 1× Wash Buffer A (Sigma-Aldrich) at room temperature. Slides were then further incubated with PLA probe solution composed of PLA plus and minus probes (Sigma-Aldrich) diluted in the Duolink antibody diluent. Incubation was carried out in a preheated humidity chamber for 1 hour at 37°C. Slides were washed twice for 5 minutes in 1× Wash Buffer A (Sigma-Aldrich, DUO82049-4L) and subjected to ligation reaction in a preheated humidity chamber for 30 minutes at 37°C, followed by washing twice with for 5 minutes in 1× Wash Buffer A (Sigma-Aldrich) at room temperature, and subjected to amplification reaction in a preheated humidity chamber in the dark for 100 minutes at 37°C. Slides were washed in the dark twice for 10 minutes in 1× Wash Buffer B (Sigma-Aldrich, DUO82049-4L), followed by a 1-minute wash in 0.01× Wash Buffer B. Slides were mounted with Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040-5ML). Slides were imaged using confocal microscopy. For quantitation, raw format confocal image files were split into individual channels by ImageJ. The green channel was subjected to thresholding. In the binary image, individual cells were selected, and spot numbers were calculated using analyze particle tool with a minimum size of 0.4 μm.

Experimental models of skin fibrosis. Complementary fibrosis models were used to evaluate the effect of RP105-KO mice from Christopher L. Karp (Cincinnati Children’s Hospital, Cincinnati, Ohio, USA) in vivo. First, 8-week-old female C57BL/6J mice received daily s.c. injections of bleomycin (10 mg/kg/d) or PBS for 2 weeks (5 days per week) and were sacrificed on day 22; lesional skin was harvested for analysis. The experiments were repeated 2 times with consistent results. In a complementary experiment, a noninflammatory fibrosis model was used. Eight-week-old female Tsk1/+ mice (C57BL/6 background, The Jackson Laboratory) were crossed with RP105-KO mice to generate RP105-KO; Tsk1/+ mice. At 12 weeks of age, RP105-KO; Tsk1/+ and Tsk/+ mice were sacrificed, and lesional skin was harvested for analysis. Six-month-old female C57BL/6J and RP105-KO mice were sacrificed, and skin was harvested for analysis (6, 36).

Paraffin-embedded tissue sections (4 μm) were stained with hematoxylin and eosin or trichrome. The thickness of the dermis and hypodermis, defined as the distance from the epidermal-dermal junctions to the dermal-adipose junction or to the loose connective tissue subjacent to the panniculus carnosus, respectively, were determined at 5 randomly selected sites/hpf (37).

For immunohistochemistry, sections of paraffin-embedded skin were immunolabeled with primary antibodies against F4/80 (Invitrogen, 1:500, 14-4801-82), CD3 (Abcam, 1:3000, ab16669), and ASMA (Abcam, 1:500, ab5694), followed by appropriate biotinylated secondary antibodies (Jackson Immunoresearch, 1:250), and detected using biotin complex conjugated with horseradish peroxidase (Vector Laboratories) and 3,3′-diaminobenzidine (DAB; Dako) for color development. The collagen content of the skin was determined by hydroxyproline assays (Biovision, Colorimetric Assay Kits). Total RNA isolated from mouse skin biopsies was reverse transcribed to cDNA using Supermix and analyzed by real-time qPCR (Applied Biosystems) on an Applied Biosystems 7500 Prism Sequence Detection System as described previously (37).

Immunofluorescence confocal microscopy using skin fibroblasts and skin biopsies. Fibroblasts seeded on 8-well Lab-Tek II chamber glass slides (Nalgene Nunc International) were incubated in serum-free DMEM with or without tenascin-C (2 μg/ml) for 72 hours. Cells were then fixed, permeabilized, and incubated with antibodies against ASMA (Abcam, 1:500, ab5694), type I collagen (Southern Biotechnology, 1:100, 1310-01), RP105 (Abcam, 1:100, ab184956), and MD1 (Santa Cruz, 1:50, sc-390613), followed by Alexa Fluor–labeled secondary antibodies (Invitrogen) as described previously (18). For tissue immunofluorescence analyses, paraffin-embedded skin sections were incubated with ASMA (Abcam, 1:100, ab5694), p-p65 (Cell Signaling Technology, 1:100, 3033), Fn-EDA (Abcam, 1:100, ab6328), tenascin-C (Abcam, 1:100, ab108930), RP105 (Abcam, 1:100, ab184956), and MD1 (Santa Cruz, 1:50, sc-390613), followed by appropriate secondary antibodies. Nuclei were detected using DAPI. Slides were mounted, and immunofluorescence and immunohistochemistry were evaluated under Nikon A1R laser scanning confocal microscope in a blinded manner. Negative controls stained without primary antibodies were used to confirm immunostaining specificity.

Second-harmonic generation. Imaging of dermal collagen fibers by second-harmonic generation was performed as previously described (38). Briefly, paraffin-embedded skin sections (12 μm thick) were scanned on a Leica SP8 microscope equipped with a Coherent Chameleon MP laser. SHG emissions were elicited using 2-photon excitation at 820 nm and detected through 410/10 nm bandwidth filters. Forward and backward SHG signals were detected by externally mounted photomultiplier tubes. Image stacks were 3D Gaussian blurred and flattened using maximum projection in ImageJ (FIJI) (39).

Statistics. Mann Whitney U test and 2-tailed Student’s t test were used for comparisons between 2 groups, with P < 0.05 considered statistically significant. Comparisons among 3 or more groups were performed using 1-way ANOVA followed by Šidák’s correction for multiple comparisons. Pearson’s rank correlations were calculated to measure the correlation. Data are presented as mean ± SD unless otherwise indicated. Data were analyzed using Graph Pad Prism (version 8).

Study approval. Biopsies were performed with written informed consent and following protocols approved by the Institutional Review Board for Human Studies at Northwestern University and the University of Michigan. Animal experiments were performed according to protocols approved by Northwestern University and the University of Michigan and in compliance with the guidelines of the Northwestern University Animal Care and Use Committee.

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