Bortezomib treatment enhances T cell apoptosis by inhibiting NF-κB activation.

<p>(<b>A</b>) The percentages of Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes 7 days after the administration of DSS. Bar graphs indicate mean (± SEM) percentages of mesenteric lymph node Annexin-V/7-AAD⁺ CD4⁺ and CD8⁺ T cells following bortezomib or control treatment in one representative experiment with 4 mice per group. (<b>B</b>) IκBα mRNA expression in mesenteric lymph nodes CD4⁺ and CD8⁺ T cells. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. A, B) Significant differences between sample means are indicated; *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Bortezomib treatment affects cytokine production in DSS-induced colits.

<p>Cytokine mRNA expression in the colon (<b>A</b>) and mesenteric lymph nodes (<b>B</b>) in mice treated with bortezomib or control during DSS-induced colitis. Colon tissue and mesenteric lymph nodes were collected from control- or bortezomib-treated mice 7 days following DSS administration. Transcript levels were quantified by real-time PCR analysis and were normalized with an internal control. Values represent means (± SEM) from ≥4 mice of each group. Significant differences between sample means are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Results represent one of two independent experiments producing similar results.</p

Bortezomib treatment reduces IFN-γ production by CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes during DSS-induced colitis.

<p>IFN-γ production by mesenteric lymph node CD4⁺ (<b>A</b>) or CD8⁺ (<b>B</b>) T cells 7 days following DSS administration as determined by intracellular cytokine staining with flow cytometry analysis. Bar graphs indicate mean (± SEM) percentages and numbers of draining lymph node IFN-γ-producing CD4⁺ or CD8⁺ T cells following bortezomib or control treatment in one representative experiment with three mice per group. Significant differences between PBS-treated mice versus other group are indicated; *<i>P</i><0.05, **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Bortezomib suppressed the severity of DSS-induced colitis.

<p>Mice ingested either DSS solution or normal drinking water. Mice were treated with 200 µl of bortezomib (0.75 mg/kg) or PBS (control) intravenously twice weekly, starting 2 days prior to DSS administration. The severity of intestinal injury was evaluated by quantitatively measuring body weight (<b>A</b>) and DAI scores (<b>B</b>). DAI scores were based on weight loss, stool consistency, and bleeding. Values represent means (±SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05; **<i>P</i><0.01. Similar results were obtained in at least two independent experiments.</p

Profile of infiltrating cells in the colon and mesenteric lymph nodes during DSS-induced colitis in mice treated with bortezomib or control.

<p>(<b>A</b>) The numbers of neutrophils, CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells, and F4/80⁺ macrophages per one field of view (×200) in the colon were counted. (<b>B</b>) Bortezomib affects CD4⁺ and CD8⁺ T cell numbers in mesenteric lymph nodes during DSS-induced colitis. Bar graphs indicate CD4⁺ T cells, CD8⁺ T cells, and B220⁺ cells in mesenteric lymph nodes 7 days after induction of colitis. A, B) Values represent means (± SEM) from ≥4 mice per group. Significant differences between sample means are indicated: *<i>P</i><0.05. Similar results were obtained in at least two independent experiments.</p

Serum pro-CTSB levels in dcSSc patients further classified into subgroups based on disease duration.

<p>dcSSc patients were divided into 3 subgroups: those with disease duration of <1 years, those with disease duration of 1 to 6 years, and those with disease duration of >6 years. Serum pro-CTSB levels were determined by a specific ELISA. The horizontal bars indicate the mean value in each group. Statistical analysis was carried out with a Kruskal-Wallis test and a Steel-Dwass' test for multiple comparison. *P<0.05.</p

CTSB expression was up-regulated in dermal vasculature of Fli1^+/− mice and in Fli1 siRNA-treated HDMECs.

<p>Immunodetection of CTSB proteins in the skin sections of 3 month-old wild type (A) and Fli1^+/− (B) mice (original magnification was ×40) by Vectastain ABC kit according to the manufacturer's instruction. Insets (original magnification was ×40) depict representative arterioles (panel 1), venules (panel 2), and capillaries (panel 3; red arrowheads), respectively. Representative results in 5 wild type and 5 Fli1^+/− mice are shown. (C) HDMECs were seeded shortly before transfection. The cells were transfected with 10 nM of Fli1 and scrambled non-silencing siRNA (Santa Cruz) using HiPerfect transfection reagent (Qiagen, Valencia, CA, USA) for 72 hours. Cells were then serum starved for the last 24 hours. mRNA levels of Fli1 and CTSB genes were examined by quantitative real-time PCR and normalized to the levels of human 18S rRNA gene. Results of controls or relative value compared with the controls are expressed as means ± SD of 3 independent experiments. Statistical analysis was carried out with a 2-tailed paired t-test. *P<0.05, **P<0.005.</p

Correlation of serum pro-cathepsin B levels with clinical features in patients with dcSSc and lcSSc.

<p>Unless noted otherwise, values are percentages. dcSSc, diffuse cutaneous systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis; MRSS, modified Rodnan total skin thickness score; DLco, diffuse capacity for carbon monoxide; VC, vital capacity; RVSP; right ventricular systolic pressure. Patients were evaluated for the presence of esophageal, pulmonary, cardiac, renal, or muscle involvements, as follows. Esophagus hypomotility was defined as distal esophageal hypomotility on barium-contrast radiography. Interstitial lung disease (ILD) was defined as bibasilar interstitial fibrosis on chest radiographs, and in patients with no abnormalities on chest radiographs early ILD was defined as alveolitis on high-resolution computer tomography. Elevated right ventricular systolic pressure (RVSP) was defined as 35 mmHg or more on echocardiogram. Cardiac involvement was defined as any of the following: symptomatic pericarditis, clinical evidence of left ventricular congestive heart failure, or arrhythmias requiring treatment. Scleroderma renal crisis was defined as malignant hypertension and/or rapidly progressive renal failure. Skeletal muscle involvement was defined as proximal muscle weakness and elevated serum creatine kinase levels, plus abnormal electromyographic findings consistent with myopathy and/or histopathologic changes in inflammatory myopathy. Disease onset was defined as the first clinical event of SSc other than Raynaud's phenomenon. Disease duration was defined as the interval between the onset and the time the blood samples were drawn. Statistical analysis was carried out with Fisher's exact probability test.</p><p>*P<0.05.</p

CTSB expression was up-regulated in dermal vasculatures of SSc patients compared to those in controls.

<p>CTSB expression levels in dermal vasculatures were determined by immunohistochemistry in skin section from 8 healthy control subjects (A, B) and 8 SSc patients (C, D). Representative results are shown. Original magnification was ×200 (A, C) and ×400 (B, D). Analysis of CTSB expression levels in vessel walls is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032272#pone-0032272-t002" target="_blank">Table 2</a>.</p

Cathepsin B levels in dermal vasculature in normal and systemic sclerosis skin.

<p>NS, normal skin; SSc, systemic sclerosis; dcSSc, diffuse cutaneous systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis. We used the following grading system: −, no staining; +, slight staining; ++, moderate staining; +++, strong staining.</p