Five patients who fulfilled diagnosis criteria for myofasciitis at Department of Neurology, Tongji Hospital were included. Biopsy specimens of muscle and fascia, as well as clinical and laboratory data obtained from these patients were retrospectively analyzed. The diagnosis of myofasciitis was determined based on suggestive clinical and laboratory findings, as well as muscle biopsy or magnetic resonance imaging (MRI) abnormalities [Table 1; (1)]. Other pathological entities, such as scleroderma, autoimmune myopathy, malignancies, and infection were excluded. There was no family history of neuromuscular disorders and no skin rashes were detected in the included patients. No vaccination was performed 1 year before the symptoms occurred. The fascia and muscle biopsy sites were determined by MRI. Muscle and fascia tissue from five patients without neuromuscular disorders served as controls. The study was approved by the Ethics Committee of Tongji Hospital and written informed consent was obtained from all included patients.

All patients underwent myositis-specific autoantibody (MAA) and myositis-associated antibody (MSA) screening using a commercial semiquantitative line blot assay (Wuhan Kindstar Diagnostics Co., Ltd., Wuhan, China). MSAs and MAAs included anti-Mi2α and β, anti-TIF1γ, anti-MDA5, anti-NXP2, anti-SAE1, anti-Jo1, anti-SRP, anti-HMGCR, anti-PL7, anti-PL12, anti-EJ, anti-OJ, anti-Ku, anti-PM/Scl-100, anti-PM/Scl-75, and anti-Ro52 antibodies. The anti-nuclear, anti-SSA/Ro60, anti-SSB/La, anti-Sm, anti-RNP, anti-mitochondrial, anti-dsDNA antibodies, and rheumatoid factors (RF) were also evaluated. Serum cytokines were assayed by electro chemiluminescence immunoassay, including interleukin-6 (IL-6) (Roche, 05109442190), tumor necrosis factor-α (TNF-α) (SIMENS, LKNF1), interleukin-2 receptor (IL-2R) (SIMENS, LKIP1), interleukin-8 (IL-8) (SIMENS, LK8P1), and interleukin-10 (IL-10) (SIMENS, LKXP1). The assessment of inflammatory indexes was performed, including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP).

All biopsy specimens were rapidly cryopreserved in liquid nitrogen, and then stored at −80°C. The frozen sections (7 μm) were stained with hematoxylin and eosin, acid phosphatase, modified Gömöri trichrome, PAS, NADH tetrazolium reductase, cytochrome C oxidase, and ATPase at pH 4.3, 4.5, and 10.4. Eosinophils were detected by Wright-Giemsa staining. The immunohistochemical (IHC) analysis of cells expressing CD3 (1:200, MAC1477, Bio-Rad), CD4 (1:50, ab133616, Abcam), CD8 (1:50, ab93278, Abcam), CD68 (1:50, ab201340, Abcam), VCAM-1 (1:200, 11,444-1-AP, Proteintech), ICAM-1 (1:200, 10,831-1-AP, Proteintech), VLA-4 (1:200, 19,676-1-AP, Proteintech), CD11a/CD18 (1:1000, ab52895, Abcam), CD31/PECAM-1 (1:2000, ab9498, Abcam), and CD146 (1:200, Ab75769, Abcam) were performed using a 3, 3′-diaminobenzidine Detection Kit; the sections were then counterstained with hematoxylin. For immunofluorescence staining, expression of major histocompatibility complex class I (MHC-I) (1:100, ab23755, Abcam) and deposition of membrane attack complex (MAC) (1:50, Sc-58,935, Santa Cruz Biotechnology) on the fascia and sarcolemma were used with Alexa Fluor conjugated secondary antibody (1:400, Invitrogen) for 1 h. Images were obtained using a microscope (Olympus) or Confocal scanning (Olympus).

A modified semiquantitative scale previously defined by Corinna Preusse was used for analysis (2). Each sample was evaluated on 20 high-power fields (HPF; one HPF as 200×). The average number of cells in 6 randomly selected HPFs per sample was calculated and scored as follows: <5 positive cells, almost no staining (−); 5–20 positive cells, sparse staining (+); 21–50 positive cells, scatter staining (++); and > 50 positive cells, abundant staining (+++). The up-regulation of MHC-I on non-necrotic myofibers and MAC deposition on sarcolemma were assessed as follows: no fibers stained (−); 0–10% of fibers stained (+); 11–30% of fibers stained (++); 31–60% of fibers stained (+++); and 61–100% of fibers stained (++++). For the positive staining on endothelium within the perimysium, the quantity and intensity were estimated separately and subjectively evaluated on a scale of -: absent, +: weak, ++: definite, +++: strong staining) (3). All biopsied sections were assessed by two independent experimenters.

Muscle and fascia tissues from controls or patients with myofasciitis were homogenized in cell lysis buffer (Beyotime, China) supplemented with protease inhibitors (MCE, China) and phosphatase inhibitors (Promoter, China). Tissue lysates were centrifuged at 15000 × g for 15 min. The supernatant was collected and protein concentration was determined using the bicinchoninic acid assay. Total protein (20–50 μg) from each sample was resolved on 8% sodium dodecyl sulfate-polyacrylamide gels and transferred to 0.45 μM nitrocellulose filter membranes (Boster, China). The membranes were blocked with 5% skim milk for 1 h at room temperature to prevent non-specific binding of antibodies and were subsequently incubated over night with the primary antibodies. Primary antibodies include those against: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, 60,004-I-Ig, Proteintech), VCAM-1 (1:1000, Ab134047, Abcam), ICAM-1 (1:1000, 10,831-1-AP, Proteintech), VLA-4 (1:1000, ab81280, Abcam), CD11a/CD18 (1:2000, ab52895, Abcam), CD31/PECAM-1 (1:2000, ab9498, Abcam), and CD146 (1:1000, Ab75769, Abcam). Membranes were washed with Tris-buffered saline with 0.05% Tween-20 and incubated for 1 h at room temperature with horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibody (1:5000, Cell Signaling Technology). Blots were developed using enhanced chemiluminescence reagents (Servicebio, China) and evaluated using a charged-coupled device camera (Tanon 4,600). Quantification of immunoreactivity was performed by densitometric analysis using Fiji (NIH) software.

The data are presented as mean ± standard deviation (SD). Mann–Whitney U test was used for two groups. The statistical significance of the differences was evaluated using GraphPad Prism (Version 8.0.1). p < 0.05 was considered statistically significant.

Five patients with myofasciitis were enrolled. The detailed clinical data of patients are presented in Table 2. Basic information of controls is displayed in Supplementary Table S1.

Limbs MRI on fat suppression T2-weighted images indicated striking abnormalities. Thigh images of Patient 1 indicated the hyperintense signal of bilateral fascia of vastus medialis muscle and semimembranosus (Figure 1A). Patchy hyperintensity was distributed in upper arm muscle groups of Patient 1. Thickness and edema of subcutaneous superficial and deep muscular fasciae on lower extremity MRI images of Patient 2 were evident (Figure 1B). Upper arms images of Patient 2 showed slight signal hyperintensity in subcutaneous fatty and perifascicular tissues. Patient 3 demonstrated that marked hyperintense signals in a perimysial and perifascicular distribution in all thigh, calf (Figure 1C), and upper limb muscle groups. Muscular edema and thickening of the fascia of the posterior thigh muscles on fat suppression T2-weighted MRI images were observed in Patient 4 (Figure 1D). Thigh (Figure 1E) and forearm images (Figure 1F) of Patient 5 showed high signal intensity of the thickened deep and superficial fascia. These manifestations were in consistent with myofasciitis in these patients.

Immunophenotype of inflammatory infiltration, up-regulation of MHC-1, and MAC deposition in biopsied specimens of patients with myofasciitis are presented in Figure 2, Table 3. Numerous inflammatory cells in the thickened fascia and perivascular inflammation were evident in biopsied specimens of patients with myofasciitis (Figures 2A-E). A few eosinophils were detected in the fascial tissues of Patient 3 but not of other four patients (Figures 2F-J). Macrophages and T lymphocytes constituted the predominant mononuclear cellular infiltrate and were dominantly distributed within the collagen tissues (Figures 2K-Y). Scatter B lymphocytes were detected in samples of all five patients (Figures 2Z-Dd). The expression of MHC-1 on the fibroblasts was particularly strong in five patients (Figures 2Ee-Ii). The intensity of MHC-I on the sarcolemma close to the immune cells was up-regulated in Patient 1 and 3. MAC was deposited on blood vessels in the fascia of the five patients (Figures 2Jj-Nn). No positive staining of CD4, CD8, CD68, and CD20 was observed in sections from controls (Supplementary Figure S1). Staining for MHC-I and MAC was detected on blood vessels in fascial tissues of control samples (Supplementary Figure S1).

The expression of CAMs in patients with myofasciitis is summarized in Table 4. Weak positive staining of VCAM-1 in the controls was noted (Figure 3A). However, there was the strong expression of VCAM-1 on vascular endothelial cells within the connective tissue and a few inflammatory cells in patients with myofasciitis (Figure 3B). No positive staining of VLA-4 was observed in the controls (Figure 3C), but there was positive staining of VLA-4 on scatter inflammatory cells in patients with myofasciitis (Figure 3D). There was low-level expression of ICAM-1 on the tissues of controls (Figure 3E) but this was up-regulated in myofasciitis (Figure 3F). CD11a/CD18 was positive on inflammatory cells (Figure 3H), whereas no positive signal was observed in controls (Figure 3G). PECAM-1 was expressed on the vascular endothelium in both controls and myofasciitis (Figures 3I,J). Light expression of CD146 on blood vessels within connective tissue in controls (Figure 3K), whereas CD146 was strongly positive in myofasciitis (Figure 3L). Western blot results were consistent with those of the immunostaining study, and showed that ICAM-1, CD11a/18, VCAM-1, VLA-4, CD31, and CD146 at the protein levels in myofasciitis were significantly increased (Figures 4A,B).