Immunopathogenesis of Behçet Disease.

BACKGROUND: Behçet's Disease (BD, OMIM 109650) is a chronic relapsing inflammatory disease of unknown etiology with unpredictable exacerbations and remissions. First described in 1937 by the Turkish dermatologist HulusiBehçet, as a trisympton complex (oral and genital ulcers and uveitis), it is now recognized as a multisystemic disease. The syndrome can manifest in diverse ways and can involve nearly every organ system. Several studies have implicated T cells and monocytes in the pathogenesis of BD especially when these cells are stimulated by heat shock proteins and streptococcal antigen. This article presents a review of the relevant published literature about the immunopathogenesis of BD. RESULT: The authors used MeSH terms "Behçet's disease" with "pathophysiology," "pathogenesis," "genetic", "epigenetic", "immunogenetic" or "immune response" to search the PubMed database. All the relevant studies identified were included.

The role of CCR1 and therapeutic effects of anti-CCL3 antibody in herpes simplex virus-induced Behçet's disease mouse model.

Behçet's disease (BD) is a chronic systemic inflammatory disease with unclear etiopathogenesis. Although gene variants of CC chemokine receptor type 1 (CCR1) have been reported, the protein expression of CCR1 in patients with BD remains unclear. The objective of this study was to analyze the frequencies of CCR1+ cells in a herpes simplex virus-induced mouse model of BD. The frequencies of CCR1+ cells on the surface and in the cytoplasm of peripheral blood mononuclear cells and lymph nodes were analyzed by flow cytometry. The CCR1+ cells were significantly down-regulated in BD mice compared with the normal control and symptom-free control mice. Colchicine and pentoxifylline treatment improved the symptoms of BD and increased the frequencies of CCR1+ cells in BD mice. Treatment with chemokine CC motif ligand 3 (CCL3), a ligand of CCR1, caused BD symptoms to deteriorate in 10 of 16 BD mice (62·5%) via down-regulation of CCR1+ cells. Anti-CCL3 antibody treatment ameliorated BD symptoms in 10 of 20 mice (50%) and significantly decreased the disease severity score compared with CCL3-treated BD mice (P = 0·01) via up-regulation of CCR1+ cell frequencies. In patients with BD, plasma levels of CCL3 in an active state were significantly higher than in healthy control individuals (P = 0·02). These results show that the up-regulation of CCR1+ cells was related to the control of systemic inflammation of BD in mouse models.

Novel Heterogeneous Mutation of TNFAIP3 in a Chinese Patient with Behçet-Like Phenotype and Persistent EBV Viremia.

PURPOSE: Tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) is a negative regulator of the nuclear factor-κB (NF-κB) pathway. It has recently been recognized that TNFAIP3 deficiency leads to early onset of autoinflammatory and autoimmune syndrome resembling Behçet's disease. Here, we report a novel mutation in TNFAIP3 in a Chinese patient, who had Behçet-like phenotype and persistent Epstein-Barr virus (EBV) viremia. METHODS: The clinical data were collected. Immunological function was detected. Gene mutation was detected by whole-exome sequencing (WES) and confirmed by Sanger sequencing. mRNA and protein levels were detected in the patient under lipopolysaccharide (LPS) stimulation by real-time PCR and Western blot. RESULTS: The patient is a 13-year-old boy, presenting with intermittent fever for 5 months, who also experienced diffuse lymphadenopathy, arthritis, and recurrent multiple gastrointestinal ulcers. EBV DNA was detected in the serum and peripheral blood mononuclear cells of the patient. The immunological phenotype showed increased proportion of double-negative T cells (CD3+CD4-CD8-). A novel missense mutation (c.1428G > A) locating at the zinc fingers 2 (ZF2) domain of TNFAIP3 inherited from his mother was confirmed. Compared with age-matched healthy controls, decrease expression of A20 was observed in the patient. The NF-κB pathway was found to be overactivated, and the synthesis of TNF-α was upregulated in the patient-derived cells. However, cells from the mother showed a milder response to LPS than cells from the patient. CONCLUSIONS: The present research indicated that the TNFAIP3 mutation of c.1428G > A (p.M476I) leads to the reduced suppression of NF-κB activation and accounted for the autoinflammatory phenotype and persistent EBV viremia in the patient.

Role of N-acetyl galactosamine-4-SO4, a ligand of CD206 in HSV-induced mouse model of Behçet's disease.

CD206 is a macrophage mannose receptor involved in variety of autoimmune and inflammatory diseases. This study aimed to identify the pathogenic role of CD206 in a herpes simplex virus (HSV) induced Behçet's disease (BD) mouse model. CD206 positive cells were detected in peripheral blood mononuclear cells and quantified by flow cytometry. Levels of cytokines were measured by ELISA. CD206 was found to be down-regulated both in vitro (10-6M) and in vivo (200µg/mouse) after treatment with N-acetylgalactosamine (GalNAc), a ligand for CD206. The down-regulation of CD206 was correlated with improvement in BD symptoms. Colchicine (2µg/mouse) or pentoxifylline (400µg/mouse) treated mice displayed improvement in BD symptoms with fewer CD206 positive cells. The prevalence of CD206-positive cells differed between ligand-responsive and non-responsive BD mice. Inhibition of CD206 was associated with down-regulated serum level of interleukin-17 in GalNAc-treated BD mice. These results suggest that the expression of CD206 is correlated with HSV-induced BD symptoms in mice, implicating that CD206 might have a pathogenic role in BD.

IL-2/IL-2 antibody immune complex regulates HSV-induced inflammation through induction of IL-2 receptor alpha, beta, and gamma in a mouse model.

The differences of serum IL-2 levels were not consistent between Behçet's Disease (BD) patients and healthy controls, however, the correlation of interleukin-2 receptor (IL-2R) and BD has not been investigated. IL-2R is composed of three subunits; alpha, beta, and gamma. The expression frequencies of IL-2R subunits were analyzed in the peripheral blood mononuclear cells, spleens, and lymph node (LN) cells. The expression of IL-2R subunits was different between BD mice and controls. IL-2R beta expressing cell frequencies were also different between BD patients and healthy controls. The IL-2/anti-mIL-2 antibody complex administration regulated the IL-2R subunits in mice. The change of expression in IL-2R was accompanied by the increase of CD8+CD44+ memory T cells, CD3-NK1.1+CD11b+CD27+ natural killer cells, and improvement of symptoms. In this study, we elucidated the role of IL-2R subunits on BD, a finding that can be connected to therapeutic strategy for patients based on the results from the treatment of BD mice.

The relevance of miRNA-21 in HSV-induced inflammation in a mouse model.

The purpose of this study was to clarify the correlation between microRNA-21 (miR-21) expression and inflammation in a herpes simplex virus (HSV)-induced Behçet's Disease (BD) mouse model. miR-21 was compared between BD patients and healthy controls in peripheral blood mononuclear cells (PBMC). For miR-21 inhibition, miR-21 antagomir was applied to BD mice. The change of symptoms was monitored. The levels of cytokines and related molecules were determined by ELISA and real time qPCR. Treatment with colchicine or pentoxifylline down-regulated the level of miR-21 with improved symptoms in mice. miR-21 inhibition was accompanied by down-regulated serum levels of IL-17 and IL-6. The expression levels of PDCD4, RhoB, PD-1, IL-12p35, and toll-like receptor-4 were also regulated by miR-21 inhibition. miR-21 was correlated with HSV-induced BD-like inflammation in mice and BD patients. The expression of miR-21 was regulated by antagomir in mice.

The role of infections in Behçet disease and neuro-Behçet syndrome.

Infections are considered an environmental trigger for exacerbations of immune-mediated diseases. We aimed to establish if common viral infections could be identified as a precipitant of Behçet disease (BD) with or without neurological involvement and to assess the variability of the immune response to common viruses. We also investigated whether cytokines and chemokines could be markers of neurological involvement. Finally, we explored if anti-basal ganglia antibodies (ABGAs) would be associated with neurological involvement in BD. Our study included 14 individuals with BD with neurological involvement (neuroBehçet syndrome - NBS), 16 individuals with BD without neurological involvement and 18 healthy controls (HC). Overall we found a tendency for increased levels of anti-viral IgG antibody levels in BD, more evident in NBS patients versus HC. Epstein-Barr viral-capsid antigen IgG titres were increased in NBS patients versus other BD patients (p=0.032). Anti-measles antibody titres induced by vaccination were similarly elevated. ABGAs were not detected in the serum of our cohort. Raised levels of serum IL-8 in some BD patients did not reflect clinical activity or severity. In conclusion, there was evidence for a polyclonal immune activation rather than a specific virus effect in the sera of individuals with BD or NBS.

Detection of the inflammatory process in a Behçet's disease-like mouse model using 18F-fluorodeoxyglucose positron emission tomography.

OBJECTIVES: The major role of herpes simplex virus (HSV) type 1 infection in Behçet's disease (BD) immunopathogenesis has been demonstrated and inoculating the earlobes of ICR mice with HSV produced a BD-like mouse model. (18)Ffluorodeoxyglucose positron emission tomography (FDG PET) is widely used for diagnosing numerous human diseases other than malignancies. The aim of our study was to evaluate the inflammatory activities of BD-like symptoms in a HSV type 1-induced BD-like mouse model by small-animal FDG PET. METHODS: Five HSV-infected ICR mice with BD-like lesions, two asymptomatic HSV-infected mice, and two untreated mice were scanned with microPET, and autopsy specimens were histopathologically assessed to evaluate for infiltration by mixed inflammatory cells. RESULTS: The histopathological evaluation of the inflammatory process in knee and elbow joints significantly correlated with the quantitative assessment of FDG accumulation in the same joints in BD-like ICR mice, HSV-infected asymptomatic mice, and untreated control mice. Small-animal FDG PET clearly detected asymptomatic joint inflammatory processes in both BD-like mice and HSV-infected asymptomatic mice. In addition, genital ulcers and skin ulcers with associated perilesional lymphadenopathies in BD-like models were detected by microPET. However, biodistributed PET-positive images from the stasis of secreted FDG into the bowel lumen could not be distinguished from the inflammatory bowel lesions of BD when compared to FDG uptake in control mice. CONCLUSIONS: Our data indicate that FDG PET can non-invasively and quantitatively detect the inflammatory process in an HSV-induced BD-like mouse model.

CCL21 attenuates HSV-induced inflammation through up-regulation of CD8+ memory cells.

CCR7 and its ligand, CCL21, are known to establish microenvironments for the initiation of immune responses in secondary lymphoid tissue. It has also been reported that CCR7 ligand gene-deleted mice have defects in lymphocyte homing. In addition, the injection of the CCR7 ligand was shown to induce the expression of memory T cells. In this study, we analyzed the expression of CCR7 and its ligand in HSV-induced Behçet's disease (BD)-like inflammation of mice. Additionally, plasmids containing the CCR7 ligand CCL19 or CCL21, pcDNA3.1-CCL19 or pcDNA3.1-CCL21, respectively, were injected into symptomatic mice, and changes in the population of memory T cells were determined. After administration of pcDNA3.1-CCL21, the frequencies of CD8+CD44+, CD8+CD62L- memory T cells were significantly up-regulated and the symptoms were not deteriorated when compared to the control vector injected group. Specifically, the difference in frequencies of CCR7+ peripheral blood mononuclear cells between active BD patients and inactive BD patients was similar to that of HSV-induced BD-like mice. These results suggest that CCR7, its ligand, and CD8+ memory cells are correlated with the regulation of BD symptoms.

Learning from HSV-infected mice as a model of Behçet's disease.

Several animal models of Behçet's disease (BD) have been proposed according to putative etiology. Among these models, the herpes simplex virus (HSV)-induced model produced the most similar disease attributes observe in patients. Inoculation of HSV type 1 to the scratched earlobe of mice produced the appropriate symptoms, including oral, genital, and skin ulcers, eye lesions, arthritis, and intestinal involvement. This HSV-induced BD model is the only continuously used model, to which various therapeutic modalities have been applied.

Galectin-9 ameliorates herpes simplex virus-induced inflammation through apoptosis.

Galectin-9 (Gal-9) has been identified as a Tim-3 ligand (L). The Tim-3-Tim-3L interaction serves as a specific down-regulator of the Th1 immune response. It has been reported that Tim-3 expression is higher in patients with inflammatory disorders such as rheumatoid arthritis compared to controls. In a herpes simplex virus-induced Behcet's disease (BD) mouse model, Tim-3 was expressed in a similarly high level. The expression of Gal-9 in macrophages from BD-like mice was lower than in asymptomatic BD normal mice; therefore, we injected 100 µg of Gal-9 into BD-like mice five times at 3 day intervals and subsequently observed changes in symptoms over 15 days. Gal-9 improved the symptoms of inflammation, decreased the severity score, and increased regulatory T cell expression in treated mice. Moreover, pro-inflammatory cytokine levels were lower in the Gal-9-treated group compared to the control group. Therefore, in the present study, Tim-3-Tim-3L interaction was found to influence inflammatory symptoms in BD-like mice.

Investigation of bacterial and viral agents and immune status in Behcet's disease patients from Iran.

AIM: Behçet's disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. METHODS: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. RESULTS: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. CONCLUSION: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

CD4(+) CD25(+) regulatory T cells ameliorate Behcet's disease-like symptoms in a mouse model.

BACKGROUND AIMS: Behcet's disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular and central nervous system involvement. It is well known that CD4(+) CD25(+) T-regulatory (Treg) cells prevent harmful immune responses to self- and non-self-antigens. In the present study, the role of Treg cells in herpes simplex virus (HSV)-induced BD-like symptoms was investigated. METHODS: HSV type 1 (F strain) inoculation of the earlobe of ICR mice has been shown to induce the development of BD-like symptoms. To determine whether the effect of Treg was associated with change in BD-like symptoms, CD4(+) CD25(+) T cells from the splenocytes of normal mice were adoptively transferred intravenously. Treg cells of splenocytes were significantly elevated following the transfer of 3 × 10(5) CD4(+) CD25(+) T cells to BD-like mice compared with the control group. RESULTS: The transfer of CD4(+) CD25(+) T cells to BD mice improved the symptoms, and the serum protein levels of interleukin (IL)-10, IL-6 and IL-17 were significantly altered compared with the control groups. Intravenous injection of anti-CD25 antibody to BD mice reduced the frequency of CD4(+) CD25(+) T cells and increased the BD severity score. We confirmed the influence of CD4(+) CD25(+) T cells on BD-like mice. CONCLUSIONS: These results show that up-regulation of the CD4(+) CD25(+) T cells in BD-like mice improves the inflammatory symptoms, while down-regulation of CD25(+) T cells is associated with deteriorated symptoms. Furthermore, these findings are correlated with changes in pro-inflammatory and anti-inflammatory cytokine levels.

Synthesized pyridine compound derivatives decreased TNF alpha and adhesion molecules and ameliorated HSV-induced inflammation in a mouse model.

Synthesized pyridine compound derivatives (SK94, SK126) from a natural lead source were administered to mice to test for possible anti-TNF alpha and anti-inflammatory activities. Lipopolysaccharide (LPS)-induced TNF alpha production was analyzed in the endothelial cells, Raw 264.7 cells, and serum of normal mice after treatment with SK compounds. These compounds were also orally administered to a herpes simplex virus (HSV)-induced Behcet's disease mouse model to investigate their anti-inflammatory therapeutic effect. TNF alpha production was inhibited in a dose-dependent manner in the SK94 treated cells. E-selectin, VCAM-1, and ICAM-1 mRNA levels were also down-regulated. Treatment with 30mg/kg SK94 inhibited 55% of the TNF alpha production in LPS challenged Balb/c mice (n=8). SK94 and SK126 were administered to the Behcet's disease-like mice for five consecutive days and SK94 improved in five out of six mice (83%), while it only improved in one out of nine mice (11%) in the pH 1.2 saline (artificial gastric juice) group (P<0.005), four out of ten mice (40%) in the thalidomide group (P<0.05), and six out of seven (86%) in the SK126 group (P<0.005). Soluble ICAM-1 was inhibited by 23.8% in the sera of SK94 treated mice and by 34.6% in SK126 treated mice when compared to artificial gastric juice. Based on these findings, SK compounds could be candidates for clinical trials.

Interleukin-6 small interfering RNA improved the herpes simplex virus-induced systemic inflammation in vivo Behcet's disease-like mouse model.

It is known that the level of interleukin-6 (IL-6) is higher in patients with active Behcet's disease (BD) than in those with inactive disease. Herpes simplex virus (HSV) type 1 inoculation of the earlobes of ICR mice resulted in the development of BD-like symptoms. To find out whether downregulation of IL-6 would affect the symptoms of BD, IL-6 small interfering RNA (siRNA) was administered to a BD mouse model. IL-6 siRNA was intraperitoneally injected into BD mice to downregulate IL-6 (n=9). IL-6 siRNA injection downregulated serum IL-6 level (118.9+/-114.4 pg ml(-1)) compared with scramble injection (439.4+/-378.0 pg ml(-1)) in BD mice (P=0.01). In seven out of nine IL-6 siRNA-injected BD mice, 77.8% improved and the severity score was decreased from 3.1+/-1.05 to 1.7+/-0.87 (P=0.005), whereas two out of six (33.3%) scramble-injected BD mice improved and the severity score changed from 2.5+/-0.84 to 2.0+/-1.41 (P=0.203). Foxp3, ROR gamma t, IL-17A, IL-17F and tumor necrosis factor-alpha were also influenced in IL-6 siRNA-injected BD mice compared with scramble-injected BD mice. Adoptive transfer of CD4+CD25+ cells to BD mice affected the decrease of IL-6 serum levels and were dependent on CD4+CD25+ cell numbers. These results showed that downregulation of IL-6 improved the inflammatory symptoms in BD mice through upregulation of regulatory T cells and inhibition of Th17 cells.

Tumor necrosis factor alpha small interfering RNA decreases herpes simplex virus-induced inflammation in a mouse model.

BACKGROUND: Anti-TNFalpha antibodies have been used for treating inflammation in patients. But, more effective and safer drugs need to be developed for improved future therapeutic use. OBJECTIVES: To inhibit the expression of TNFalpha, we used small interfering RNAs (siRNAs) to reduce over expression of TNFalpha in vitro in cell cultures and in an in vivo Behcet's disease-like (BD) mouse model for amelioration of chronic inflammation. METHODS: TNFalpha siRNA was injected intraperitoneally twice with a 1-week interval. To compare the efficacy of TNFalpha siRNA versus an anti-TNFalpha antibody, Infliximab and Etanercept were administered to symptomatic mice with inflamed tissue. RESULTS: Intraperitoneal delivery of TNFalpha siRNA effectively decreased BD symptoms in 18 of 32 cases (56.3%). Scrambled siRNA treatment decreased BD symptoms in 2 of 19 cases (10.5%). Infliximab was effective in 11 of 27 cases (40.7%) and Etanercept was also effective in 9 of 25 cases (36.0%) at the end of the second week after treatment. TNFalpha siRNA reduced serum levels of TNFalpha (1.57 +/- 0.43pg/ml), compared to levels in mice not injected (84.02 +/- 24.59pg/ml) (p<0.01) or scramble injected (118.89 +/- 20.08pg/ml) (p<0.01). After single injection of TNFalpha siRNA, improvement of BD symptoms showed at 9 +/- 7th day on an average, contrary, in Infliximab injected group, improvement was apparent at 15 +/- 4th day after injection (p<0.05). CONCLUSION: We show that siRNAs can be employed to inhibit cytokine gene expression in an in vivo disease mouse model. This inhibition may, therefore, be attributed to the improvement of inflammatory symptoms.

Detection of parvovirus B19 DNA in the lesional skin of patients with Behçet's disease.

BACKGROUND: There is disagreement in the current evidence for viral aetiologies in the pathogenesis of Behçet's disease (BD). OBJECTIVES: To investigate the presence of B19 DNA in skin lesions of patients with BD, compare with the skin of healthy controls and evaluate its role in the pathogenesis. METHODS: In total, 40 patients diagnosed with BD according to the criteria proposed by the International Study Group for Behçet's Disease and routinely followed up at our centre were enrolled into the study. All the patients selected were in the active phase of disease. Skin and blood samples of patients with BD and of the healthy volunteers were examined for B19 serology, histopathology and genome expression. RESULTS: The quantity of B19 DNA in nonulcerative BD lesions of was significantly different from ulcerative lesions in the study group and from the skin of the healthy controls (P < 0.01). For the nonulcerative lesions, real-time PCR analysis for B19 DNA was found to be 64% sensitive (95% CI 42.5-82.0) and 85% specific (95% CI 62.1-96.6) with a cut-off value of > 154 IU/mL (P < 0.001). CONCLUSIONS: To the best of our knowledge, this is the first study that provides evidence for a possible causal link between BD and parvovirus B19, and our data suggest the presence of the virus, particularly in intact, nonulcerative skin lesions of BD. Limitations to this study include the limited number of participants, and the fact that the exact source of B19 DNA was undetected.