A 69-year old man was admitted for further examination of bloody diarrhea, appetite loss, and high-grade fever (>38 C), that persisted ~1 month. He was diagnosed with uveitis and treated at the Department of Ophthalmology. On admission, he developed periodic fever accompanied by bloody diarrhea. Physical examination revealed oral aphthous ulcers without genital ulcers or erythema nodosum. Leukocytosis (white blood cell count: 10,200/muL) and anemia (hemoglobin: 9.1 g/dL) were detected in complete blood cell counts. Although, the liver and kidney function parameters were within normal limits, severe hypoalbuminemia (albumin: 1.7 g/dL, normal range 4.1-5.1) was seen. Serum concentrations of C-reactive protein (8.717 mg/dL, normal range <0.14) and IgG (2,492 mg/dL, normal range 861-1,747) were elevated. The patient's human leukocyte antigen was B54, and neither anti-neutrophil cytoplasmic antibody nor anti-nuclear antibody was positive. No pathogenic bacteria were detected in blood or stool cultures.
Whole body computed tomography was performed to identify the causes of bloody diarrhea and periodic fever. Diffuse wall thickening of the ascending and transverse colon was detected on abdominal computed tomography. Deep, punched-out, oval-shaped, and semi-circumferential ulcers were observed in the cecum and sigmoid colon, respectively, during colonoscopy (Figure 1A). Pathological examination revealed destruction of the crypt architecture and massive accumulation of lymphocytes and granulocytes in the colonic lamina propria (Figure 1B). Granuloma formation or crypt abscesses were not observed. Polymerase chain reaction (PCR) tests for the detection of tuberculosis or cytomegalovirus using colonic biopsy samples were negative. These endoscopic and pathological findings were not consistent with Crohn's disease or ulcerative colitis. Based on the presence of uveitis, oral ulcers, and intestinal symptoms, the patient was diagnosed with intestinal BD according to the international diagnostic criteria for BD.
Colonic biopsy samples were subjected to immunohistochemical analysis, as previously described. Briefly, deparaffinized sections were incubated with rabbit anti-human CD3 monoclonal antibody (mAb, clone 2GV6, Roche Diagnostics), mouse anti-human CD20 mAb (clone L26, Roche Diagnostics), rabbit anti-human polyclonal myeloperoxidase (MPO)+ Ab (#518-114428, Roche Diagnostics) and mouse anti-human CD68 mAb (clone KP-1, Agilent Technology) followed by visualization of protein expression by Dako EnVision+ System (Agilent Technology). Primary Ab dilution was done according to the recommendation of the manufacturers. Immunohistochemical photographs were captured by microscopy (Biozero, BZ-8100, Keyence). As shown in Figure 1B, the colonic mucosa was characterized by massive accumulation of CD3+ T cells, CD68+ macrophages, and MPO+ granulocytes, but not of CD20+ B cells (data not shown).
The initial treatment with oral colchicine (1.5 mg/day) based on the diagnosis of intestinal BD was unsuccessful. The patient was then treated with oral PSL (40 mg/day) with a tapering schedule as shown in the clinical course (Figure 2A), which again failed to improve his symptoms. As shown in Figure 2B (top panel), active ulcers were still present in the cecum and sigmoid colon. IFX (5 mg/kg) was then administered to induce remission. Five injections of IFX, which was tolerable without adverse events, led to complete remission in this case, as indicated by the complete disappearance of colonic ulcers through colonoscopy, and by improvement of the patient's symptoms (Figure 2B, bottom panel). Thus, this case of intestinal BD was successfully treated with IFX.
Total mRNA was isolated from colonic biopsy samples during colonoscopy and subjected to quantitative PCR (qPCR) analyses as previously described. mRNA was isolated from colonic biopsy samples by using Monarch Total RNA Miniprep kit (New England Biolabs) and then was reverse transcribed into cDNA by High-Capacity RNA to cDNA kit (Applied Biosystems). The mRNA level for each target gene was determined by SYBR Green-based qPCR using a Step One Plus system (Applied Biosystems) and Quantitect primer assays (Qiagen) followed by the normalization using beta-actin as a reference gene. Each PCR primer for Quantitect primer assays was purchased from Qiagen as follows; beta-actin (#QT00095431), TNF-alpha (#QT00029162), IFN-beta (#QT000203763), IFN-gamma (#QT00000525), IL-6 (#QT00083720), IL-9 (#QT00000630), IL-12 (#QT00000364), IL-13 (#QT00000511), IL-33 (#QT00041559), C-C chemokine ligand 17 (CCL17, #QT00096866), CCL26 (#QT00023135), FOXP3 (#QT00048286), C-X-C motif chemokine ligand 9 (CXCL9, #QT00013461), and CXCL10 (#QT01003065). In some experiments, custom made primers were obtained from Invitrogen. Primer sequences were as follows; IL-1beta (sense; GGACAAGCTGAGGAAGATGC, antisense; TCGTTATCCCATGTGTCGAA), IL-5 (sense; TGAGGATGCTTCTGCATTTG, antisense; GCAGTGCCAAGGTCTCTTTC), IL-17 (sense; CATGAACTCTGTCCCCATCC, antisense; CCCACGGACACCAGTATCTT), IL-23 (sense; TCTCCTTCTCCGCTTCAAAA, antisense; TTAGGGACTCAGGGTTGCTG). Quality of RNA and cDNA was checked by NanoDrop (Thermo Fisher Scientific). Annealing temperature was optimized and the specificity of the reaction was confirmed by the presence of a sharp single peak in melting curve analyses. mRNA expression levels of target genes were normalized by that of beta-actin mRNA by using Ct method. Thus, qPCR analyses were performed in accordance with the MIQE guidelines.
The levels of mRNAs encoding proinflammatory cytokines and chemokines were compared in samples isolated before treatment (Day 7), after treatment with PSL (Day 42), and after three injections of IFX (Day 98). qPCR analyses revealed that the colonic mucosa before treatment was characterized by elevated mRNA expression levels of IL-17, IL-23, CXCL9, CXCL10, and IFN-beta (Figure 3A). Although, treatment with PSL led to a marked decrease in all proinflammatory cytokines and chemokines at mRNA level, remission was not achieved. Notably, successful induction of remission by IFX was accompanied by elevated mRNA expression levels of CXCL9, CXCL10, IFN-beta, IFN-gamma, IL-1beta, IL-6, and IL-33 as compared with those after PSL treatment. IL-6 mRNA expression was particularly strongly induced by IFX, suggesting that IL-6 may be involved in the improvement of chronic inflammation in this case. Expression of colitogenic pro-inflammatory cytokines at mRNA level, such as IL-17, IL-23, and TNF-alpha were suppressed by both PSL and IFX treatments, compared to their levels before treatment. We also examined the mRNA expression of FOXP3 and found that treatment with IFX markedly increased its levels in the colonic mucosa (Figure 3B). Thus, increased expression of IL-6 and FOXP3 and decreased expression of TNF-alpha at mRNA level might be key factors in the resolution of severe inflammation in this case of intestinal BD.