Single-cell analyses highlight the proinflammatory contribution of C1q-high monocytes to Behçet's disease.

Behçet's disease (BD) is a chronic vasculitis characterized by systemic immune aberrations. However, a comprehensive understanding of immune disturbances in BD and how they contribute to BD pathogenesis is lacking. Here, we performed single-cell and bulk RNA sequencing to profile peripheral blood mononuclear cells (PBMCs) and isolated monocytes from BD patients and healthy donors. We observed prominent expansion and transcriptional changes in monocytes in PBMCs from BD patients. Deciphering the monocyte heterogeneity revealed the accumulation of C1q-high (C1qhi) monocytes in BD. Pseudotime inference indicated that BD monocytes markedly shifted their differentiation toward inflammation-accompanied and C1qhi monocyte-ended trajectory. Further experiments showed that C1qhi monocytes enhanced phagocytosis and proinflammatory cytokine secretion, and multiplatform analyses revealed the significant clinical relevance of this subtype. Mechanistically, C1qhi monocytes were induced by activated interferon-γ (IFN-γ) signaling in BD patients and were decreased by tofacitinib treatment. Our study illustrates the BD immune landscape and the unrecognized contribution of C1qhi monocytes to BD hyperinflammation, showing their potential as therapeutic targets and clinical assessment indexes.

Baricitinib for the treatment of refractory vascular Behçet's disease.

OBJECTIVE: The pilot study aims to evaluate the effectiveness and safety of baricitinib in Behcet's Disease (BD) patients with refractory vascular involvement. METHODS: We consecutively enrolled vascular/cardiac BD patients who received baricitinib (2 mg/day) along with glucocorticoids (GCs) and immunosuppressants in our center. Efficacy assessment mainly depends on the proportion of clinical remission and side effects were recorded. RESULTS: 17 patients (12 males) were included with a mean follow-up of 10.7 ± 5.3 months. At 3 months of follow-up, 76.5% of patients achieved a complete response and the proportion increased to 88.2% at the last visit. During follow-up, ESR (p < 0.01) and hsCRP (p < 0.0001) decreased significantly, as well as Behçet's Disease Current Activity Form score (p < 0.01). In addition, baricitinib showed a GCs-sparing effect. No serious adverse events were noted. CONCLUSIONS: Our study suggests that baricitinib is well-tolerated and effective in treating refractory vascular/cardiac BD patients.

Baricitinib for the treatment of intestinal Behçet's disease: A pilot study.

OBJECTIVES: The pilot study aims to explore the efficacy and safety of baricitinib in treating refractory intestinal Behçet's disease (BD). METHODS: We consecutively enrolled patients with refractory intestinal BD from October 2020 to September 2022. They were treated with baricitinib 2-4 mg daily, with background glucocorticoids and immunosuppressants. Efficacy assessment included the global gastrointestinal symptom scores, the endoscopy scores, the Disease activity index for intestinal Behçet's disease (DAIBD), and the inflammatory parameters. Side effects were recorded. RESULTS: The thirteen patients (six males and seven females) had a median follow-up of eleven months, 76.92% (10/13) patients achieved complete remission of global gastrointestinal symptom scores, and 66.7% (6/9) had mucosal healing on endoscopy. The DAIBD scores decreased significantly, as well as the C-reactive protein level. Baricitinib showed a glucocorticoid-sparing effect, and the safety profile is favorable. CONCLUSION: Baricitinib might be a potential choice in treating refractory intestinal BD.

The clinical features, image findings and risk factors of vena cava syndrome in Behçet's syndrome.

OBJECTIVES: To investigate the clinical features, image findings, and potential risk factors of vena cava syndrome (VCS) in Behçet's syndrome (BS). METHODS: We conducted a case-control study in our BS registry database from 2012 to 2021. Fifty-five BS patients with VCS were enrolled in the case group, and two BS patients without VCS were selected as controls for each VCS case using risk-set-sampling. Multivariable logistic regression was used to detect the risk factors of VCS, and the outcome of these patients was also analysed. We also conducted an exploratory study to evaluate spectral computed tomography (CT) imaging in detecting thrombosis in BS patients with inferior VCS (IVCS). RESULTS: Multivariable logistic regression analysis revealed male gender (OR 11.16, 95%CI 3.34-37.32), early-onset BS (<18 years) (OR 5.57, 95%CI 1.58-19.69), ESR >60 mm/hr (OR 3.83, 95%CI 1.02-12.23) and pathergy reaction (OR 5.10, 95%CI 2.11-12.32) as potential risk factors of VCS in BS patients. For 4 BS patients with IVCS due to thrombosis, spectral CT found better contrast between IVC and thrombi at a low energy level of 40keV using virtual monoenergetic imaging than conventional images at 120kV. With a median follow-up of 3.3 years, the respective estimated 1- and 5-year survival rates were 96.3% and 94.2%, and respective estimated 1- and 5-year relapse-free rates were 93.9% and 78.0%. CONCLUSIONS: Male, early-onset BS, ESR >60 mm/hr, and pathergy reaction are potential risk factors of VCS in BS patients. Spectral CT is valuable in detecting thrombus in vena cava.

Aberrant monocyte subsets in patients with Behçet's disease.

Monocytes are a versatile and dynamic population, but its implication in Behçet's Disease (BD) remains elusive. We investigated the proportion, phenotypical and functional alterations in classical monocytes (CM), intermediate monocytes (IM) and non-classical monocytes (NCM) subsets in BD. A higher IM and lower NCM population in BD patients were noted, closely correlated with disease activity, and rescued after treatment. CD11b and CD64 expression on CM, IM and NCM in BD were enhanced. BD CM promoted TNF-a and IL-6 production and facilitated Th1 differentiation. BD IM promoted IL-6 production. We further demonstrated a higher level of phosphorylated p65 (p-p65) in BD CM and increased p-p65 and p-p38 in BD IM. Our data highlighted the aberrant populations of IM and CM in BD, potentially implicated in BD' pathogenesis.

Proteomics Landscape Mapping of Organ-Resolved Behçet's Disease Using In-Depth Plasma Proteomics for Identifying Hyaluronic Binding Protein 2 Expression Associated With Vascular Involvement.

OBJECTIVE: This study was undertaken to elucidate the pathogenesis and heterogeneity of Behçet's disease (BD) involving different organs using in-depth proteomics to identify the biomarkers for clinical assessment and treatment of patients with BD. METHODS: We measured the expression levels of proteins in plasma samples from 98 patients with BD and from 31 healthy controls using our in-depth proteomics platform with a data-independent acquisition mass spectrometer and antibody microarray. We performed bioinformatics analyses of the biologic processes and signaling pathways that were changed in the BD group and constructed a proteomics landscape of organ-resolved BD pathogenesis. We then validated the biomarkers of disease severity and the vascular subset in an independent cohort of 108 BD patients and 29 healthy controls using an enzyme-linked immunosorbent assay. RESULTS: The BD group had 220 differentially expressed proteins, which discriminated between BD patients (88.6%) and healthy controls (95.5%). The bioinformatics analyses revealed different biologic processes associated with BD pathogeneses, including complement activation, wound healing, angiogenesis, and leukocyte-mediated immunity. Furthermore, the constructed proteomics landscape of organ-resolved BD identified proteomics features of BD associated with different organs and protein targets that could be used for the development of therapeutic treatment. Hyaluronic binding protein 2, tenascin, and serpin A3 were validated as potential biomarkers for the clinical assessment of vascular BD and treatment targets. CONCLUSION: Our results provide valuable insight into the pathogenesis of organ-resolved BD in terms of proteomics characteristics and potential biomarkers for clinical assessment and potential therapies for vascular BD.

Macrophage polarization toward M1 phenotype through NF-κB signaling in patients with Behçet's disease.

BACKGROUND: Macrophages are key innate immune cells implicated in the pathogenesis of Behçet's disease (BD), and macrophage polarization plays a pivotal role in inflammatory response. This study aimed to investigate the role of BD serum on the phenotypes and functions of macrophage polarization. METHODS: BD or HC serum-treated human monocyte-derived macrophages (HMDMs) were examined M1/M2 phenotypes using flow cytometry and ELISA. The phagocytic capacity of HMDMs and CD4+T cell differentiation facilitated by HMDMs were measured by flow cytometry. Transcriptome analysis of BD and HC serum-stimulated HMDMs was conducted to identify differentially expressed genes. NF-κB signaling was examined using western blot to explore the mechanism of macrophage polarization induced by BD serum. RESULTS: BD serum-treated macrophages expressed a higher level of CD86, IL-12, and TNF-α and a lower level of CD163, which were compatible with the M1-like phenotype. Furthermore, BD serum-treated macrophages showed enhanced phagocytic capacity and promoted more Th1 cell differentiation. Sixty-one differentially expressed genes were identified between BD and HC serum-treated macrophages and were enriched in NF-κB signaling. BD serum-treated macrophages showed upregulated p-p65 and downregulated IκBα, and NF-κB inhibitor attenuated BD serum-stimulated M1-like phenotype. CONCLUSIONS: BD serum promoted macrophage polarization toward a proinflammatory M1-like phenotype through NF-κB signaling and potentially facilitated inflammation in BD. M1 polarized macrophages may be a potential therapeutic target for BD.

Effectiveness and safety of low-dose interferon alpha-2a treatment in Behçet's Syndrome with refractory vascular or neurological involvement: a case series.

Objective: The aim of this study was to evaluate the effectiveness and safety of low-dose interferon alpha-2a (IFNα2a) in Behçet's syndrome (BS) patients with refractory vascular/cardiac or neurological involvement. Methods: In this retrospective cohort study, we consecutively included 25 BS patients with refractory vascular/cardiac (n = 16) or neurological involvement (n = 9) who received IFNα2a treatment in our center between June 2018 and September 2021. The low-dose IFNα2a (3 million IU, every other day) was used as an add-on treatment with the continuation of glucocorticoids (GCs) and immunosuppressants. Results: In total, 25 patients (20 males, 5 females) with a mean age of 31.92 ± 9.25 years were included. IFNα2a was administered for BS patients with refractory vascular/cardiac involvement (n = 16) and neurological involvement (n = 9). Before the initiation of IFNα2a, patients had insufficient response or intolerance to conventional therapies. After a median follow-up of 23 [interquartile range (IQR), 11-30] months, all patients achieved clinical improvement. The Behçet's disease Current Activity Form (BDCAF) score improved significantly (5 versus 0, median, p < 0.0001). BS Overall Damage Index (BODI) and vasculitis damage index (VDI) remain stable (p > 0.05). Decrease in erythrocyte sedimentation rate [ESR; 24 (IQR, 12-43.5) versus 5 (IQR, 2.75-10.5) mm/h, p = 0.0001] and C-reactive protein [CRP; 6.64 (IQR, 3.67-19.82) versus 1.24 (IQR, 0.24-3.12) mg/liter, p < 0.005] was achieved effectively. The median GCs dosage tapered from 26.25 (IQR, 11.88-41.25) to 10.00 (IQR, 7.50-10.63) mg/d, p < 0.0001. Immunosuppressants were also reduced in number (p < 0.005). No serious adverse events were observed during follow-up. Conclusion: Our study suggests that low-dose IFNα2a, combined with GCs and immunosuppressants, is well-tolerated and effective for BS patients with refractory vascular/cardiac or neurological involvement and has a steroid- and immunosuppressant-sparing effect.

Perioperative management with biologics on severe aortic valve regurgitation caused by Behçet syndrome: the experience from a single center.

BACKGROUND: To investigate the efficacy and safety of biologics in the perioperative management of severe aortic valve regurgitation (AR) caused by Behçet syndrome (BS). METHODS: We retrospectively analyzed 20 patients with severe AR caused by BS who were all treated with biologics during the perioperative period of cardiac surgeries in our center between February 2016 and October 2020. RESULTS: A total of 20 patients with severe AR were enrolled, including 19 males and 1 female, with a mean age of 39.1 ± 8.8 years and a median course of 8 [interquartile range (IQR) 5.25-10.00] years. Before biologic administration, 92.9% of the patients who underwent aortic valve replacement had failed conventional therapy and developed postoperative paravalvular leakage (PVL) at a median interval of 4 months. Biologics were administered with background glucocorticoids (GCs) and immunosuppressants during the perioperative period for 22 aortic valve surgeries, including preoperatively with a median interval of 3.5 (IQR 2.75-4.25) months in 13 cases and within 3 months postoperatively in 9 cases. After a median follow up of 21 (IQR 15-32) months, 2 out of 13 cases (15.4%) preoperatively, and 1 out of 9 cases (11.1%) postoperatively treated with biologics developed PVL, and the rest were event free. The Behçet's Disease Current Activity Form score improved significantly (7 versus 0, median, p < 0.0001). Decrease of erythrocyte sedimentation rate [25.0 (IQR 11.00-36.25) mm/h versus 6.5 (IQR 4.0-8.8) mm/h, p < 0.001], and C-reactive protein [20.77 (IQR 7.19-29.58) mg/l versus 1.53 (IQR 0.94-2.92) mg/l, p = 0.001] were achieved rapidly and effectively. The GC dosage tapered from 40 (IQR 30-60) mg/d to 10 (IQR 5-11.25) mg/d, p < 0.0001. Immunosuppressants were tapered in number and dosage in 6 (30%) and 20 patients (100%), respectively. No serious adverse event was observed. CONCLUSION: Our study suggests that biologics were effective and well tolerated for the perioperative management of severe and refractory AR caused by BS, which significantly reduced the occurrence of postoperative PVL and had favorable GC- and immunosuppressant-sparing effect.

Tocilizumab in the treatment of severe and refractory parenchymal neuro-Behçet's syndrome: case series and literature review.

OBJECTIVES: This study aimed to investigate the efficacy and safety of tocilizumab (TCZ) in severe and refractory parenchymal neuro-Behçet's syndrome (p-NBS). METHODS: We retrospectively analyzed five patients with p-NBS treated with TCZ in our center between 2013 and 2020, and six cases from literature research with the index terms "neuro-Behçet's syndrome" and "tocilizumab" on PubMed NCBI. RESULTS: A total of 11 patients with p-NBS were enrolled (5 males, 6 females), with a mean age of 34.5 ± 8.0 years at the onset. All the patients had parenchymal neurological lesions, six patients (54.5%) suffered from multiple lesions, and nine patients (81.8%) were disabled. Before TCZ administration, all the patients had failed conventional therapy, eight patients (72.7%) received two or more immunosuppressants, and five patients showed insufficient response or intolerance to other biologics. TCZ was administrated at 8 mg/kg every 4 weeks, with background glucocorticoids (GCs) and immunosuppressants. After a median follow-up of 13 (interquartile range, 3.5-23.5) months, all the patients achieved both clinical and radiological improvements, and the Behçet's Disease Current Activity Form score improved significantly (3 versus 0, median, p = 0.004), the Rankin score also decreased (4 versus 2, median, p = 0.005). Levels of interleukin-6 in the cerebrospinal fluid decreased significantly in five patients (533.4 ± 389.7 pg/ml versus 34.5 ± 27.1 pg/ml, p = 0.048), after a median of two (interquartile range, 1-4) times of TCZ infusions. Furthermore, the GC dosage (per os) reduced from 69.2 ± 16.9 mg/d to 16.4 ± 16.2 mg/d (p = 0.000), and immunosuppressants were tapered in number and dosage in seven (63.6%) and four (36.3%) patients, respectively. No serious adverse events or deaths were observed during follow-up. CONCLUSIONS: TCZ is well tolerated and effective in severe and refractory p-NBS, with a favorable GC- and immunosuppressant-sparing effect. Cerebrospinal fluid interleukin-6 might be used to monitor the effects of TCZ in p-NBS.

Neutrophil Extracellular Traps Promote Aberrant Macrophages Activation in Behçet's Disease.

Neutrophil extracellular traps (NETs) are upregulated and promote thrombosis in Behçet's disease (BD). However, whether NETs promote autoinflammation in BD remains unclear. This study aimed to investigate the potential role of NETs in promoting macrophage activation in BD. Firstly, we quantified NETs by measuring double-stranded DNA (dsDNA) using PicoGreen and calculating the proportion of NETosis. Then macrophages were stimulated with BD- or healthy controls (HC)-derived NETs, and IL-8 and TNF-α production and IFN-γ+ CD4+ T cells differentiation were measured using ELISA and flow cytometry, respectively. The protein components in NETs were analyzed by western blot. Macrophages were stimulated with Histone H4 neutralized NETs, and IL-8 and TNF-α production were measured using ELISA. The level of 8-hydroxydeoxyguanosine (8-OHdG) DNA in NETs was measured using ELISA. The levels of reactive oxygen species (ROS) in serum and neutrophils were measured using ROS probes by a microplate reader and flow cytometry. We found that circulating NETs and neutrophil-derived NETs were significantly higher in BD than HC. BD NETs stimulated macrophages produced higher levels of IL-8 and TNF-α, and promoted IFN-γ+ CD4+ T cells differentiation. BD NETs were enriched in Histone H4, and neutralizing Histone H4 abrogated the BD NETs-mediated IL-8 production by macrophages, but not TNF-α. Also, BD neutrophils produced more 8-OHdG DNA than HC neutrophils, and the percentage of 8-OHdG DNA in dsDNA from BD neutrophils was also higher than that of HC neutrophils. The ROS levels in serum and neutrophils were both higher in BD than HC. Our findings suggested that excessive BD NETs promoted macrophages activation and facilitated IFN-γ+ CD4+ T cells differentiation. Higher levels of Histone H4 and oxidized DNA in BD NETs might mediate macrophages hyperactivation.