Single-cell profiling reveals kidney CD163+ dendritic cell participation in human lupus nephritis.

OBJECTIVES: The current work aimed to provide a comprehensive single-cell landscape of lupus nephritis (LN) kidneys, including immune and non-immune cells, identify disease-associated cell populations and unravel their participation within the kidney microenvironment. METHODS: Single-cell RNA and T cell receptor sequencing were performed on renal biopsy tissues from 40 patients with LN and 6 healthy donors as controls. Matched peripheral blood samples from seven LN patients were also sequenced. Multiplex immunohistochemical analysis was performed on an independent cohort of 60 patients and validated using flow cytometric characterisation of human kidney tissues and in vitro assays. RESULTS: We uncovered a notable enrichment of CD163+ dendritic cells (DC3s) in LN kidneys, which exhibited a positive correlation with the severity of LN. In contrast to their counterparts in blood, DC3s in LN kidney displayed activated and highly proinflammatory phenotype. DC3s showed strong interactions with CD4+ T cells, contributing to intrarenal T cell clonal expansion, activation of CD4+ effector T cell and polarisation towards Th1/Th17. Injured proximal tubular epithelial cells (iPTECs) may orchestrate DC3 activation, adhesion and recruitment within the LN kidneys. In cultures, blood DC3s treated with iPTECs acquired distinct capabilities to polarise Th1/Th17 cells. Remarkably, the enumeration of kidney DC3s might be a potential biomarker for induction treatment response in LN patients. CONCLUSION: The intricate interplay involving DC3s, T cells and tubular epithelial cells within kidneys may substantially contribute to LN pathogenesis. The enumeration of renal DC3 holds potential as a valuable stratification feature for guiding LN patient treatment decisions in clinical practice.

Targeted therapies for lupus nephritis: Current perspectives and future directions.

ABSTRACT: Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus, poses a substantial risk of progression to end-stage renal disease, with increased mortality. Conventional therapy for LN relies on broad-spectrum immunosuppressants such as glucocorticoids, mycophenolate mofetil, and calcineurin inhibitors. Although therapeutic regimens have evolved over the years, they have inherent limitations, including non-specific targeting, substantial adverse effects, high relapse rates, and prolonged maintenance and remission courses. These drawbacks underscore the need for targeted therapeutic strategies for LN. Recent advancements in our understanding of LN pathogenesis have led to the identification of novel therapeutic targets and the emergence of biological agents and small-molecule inhibitors with improved specificity and reduced toxicity. This review provides an overview of the current evidence on targeted therapies for LN, elucidates the biological mechanisms of responses and failure, highlights the challenges ahead, and outlines strategies for subsequent clinical trials and integrated immunomodulatory approaches.

Cutting Edge: Serpine1 Negatively Regulates Th1 Cell Responses in Experimental Autoimmune Encephalomyelitis.

Th1 cells are critical in experimental autoimmune encephalomyelitis (EAE). Serine protease inhibitor clade E1 (Serpine1) has been posited as an inhibitor of IFN-γ from T cells, although its role in autoimmunity remains unclear. In this study, we show that Serpine1 knockout (KO) mice develop EAE of enhanced severity relative to wild-type (WT) controls. Serpine1 overexpression represses Th1 cell cytokine production and pathogenicity, whereas Serpine1-KO:2D2 Th1 cells transfer EAE of increased severity in comparison with WT 2D2 Th1 cells. Notably, polarized Serpine1-KO Th1 cells display delayed expression of the Th1-specific inhibitory receptor, Tim-3 (T cell Ig and mucin-domain containing-3). Serpine1-KO:Tim-3-Tg Th1 cells, which transgenically overexpress Tim-3, showed increased expression of IFN-γ and reduced expression of the checkpoint molecules Lag-3 and PD-1 relative to WT Tim-3-Tg counterparts. Furthermore, Serpine1 deficiency restored the EAE phenotype of Tim-3-Tg mice that normally develop mild disease. Taken together, we identify Serpine1 as a negative regulator of Th1 cells.

Group 3 Innate Lymphoid Cells Exacerbate Lupus Nephritis by Promoting B Cell Activation in Kidney Ectopic Lymphoid Structures.

Group 3 innate lymphoid cells (ILC3s) represent a new population in immune regulation, yet their role in lupus nephritis (LN) remains elusive. In the present work, systemic increases in ILC3s, particularly in the kidney, are observed to correlate strongly with disease severity in both human and murine LN. Using MRL/lpr lupus mice and a nephrotoxic serum-induced LN model, this study demonstrates that ILC3s accumulated in the kidney migrate predominantly from the intestine. Furthermore, intestinal ILC3s accelerate LN progression, manifested by exacerbated autoimmunity and kidney injuries. In LN kidneys, ILC3s are located adjacent to B cells within ectopic lymphoid structures (ELS), directly activating B cell differentiation into plasma cells and antibody production in a Delta-like1 (DLL1)/Notch-dependent manner. Blocking DLL1 attenuates ILC3s' effects and protects against LN. Altogether, these findings reveal a novel pathogenic role of ILC3s in B cell activation, renal ELS formation and autoimmune injuries during LN, shedding light on the therapeutic value of targeting ILC3s for LN.

Monogenic lupus: Tracing the therapeutic implications from single gene mutations.

Monogenic lupus, a distinctive variant of systemic lupus erythematosus (SLE), is characterized by early onset, family-centric clustering, and heightened disease severity. So far, over thirty genetic variations have been identified as single-gene etiology of SLE and lupus-like phenotypes. The critical role of these gene mutations in disrupting various immune pathways is increasingly recognized. In particular, single gene mutation-driven dysfunction within the innate immunity, notably deficiencies in the complement system, impedes the degradation of free nucleic acid and immune complexes, thereby promoting activation of innate immune cells. The accumulation of these components in various tissues and organs creates a pro-inflammatory microenvironment, characterized by a surge in pro-inflammatory cytokines, chemokines, reactive oxygen species, and type I interferons. Concurrently, single gene mutation-associated defects in the adaptive immune system give rise to the emergence of autoreactive T cells, hyperactivated B cells and plasma cells. The ensuing spectrum of cytokines and autoimmune antibodies drives systemic disease manifestations, primarily including kidney, skin and central nervous system-related phenotypes. This review provides a thorough overview of the single gene mutations and potential consequent immune dysregulations in monogenic lupus, elucidating the pathogenic mechanisms of monogenic lupus. Furthermore, it discusses the recent advances made in the therapeutic interventions for monogenic lupus.

Tim-3 adapter protein Bat3 acts as an endogenous regulator of tolerogenic dendritic cell function.

Dendritic cells (DCs) sense environmental cues and adopt either an immune-stimulatory or regulatory phenotype, thereby fine-tuning immune responses. Identifying endogenous regulators that determine DC function can thus inform the development of therapeutic strategies for modulating the immune response in different disease contexts. Tim-3 plays an important role in regulating immune responses by inhibiting the activation status and the T cell priming ability of DC in the setting of cancer. Bat3 is an adaptor protein that binds to the tail of Tim-3; therefore, we studied its role in regulating the functional status of DCs. In murine models of autoimmunity (experimental autoimmune encephalomyelitis) and cancer (MC38-OVA-implanted tumor), lack of Bat3 expression in DCs alters the T cell compartment-it decreases TH1, TH17 and cytotoxic effector cells, increases regulatory T cells, and exhausted CD8+ tumor-infiltrating lymphocytes, resulting in the attenuation of autoimmunity and acceleration of tumor growth. We found that Bat3 expression levels were differentially regulated by activating versus inhibitory stimuli in DCs, indicating a role for Bat3 in the functional calibration of DC phenotypes. Mechanistically, loss of Bat3 in DCs led to hyperactive unfolded protein response and redirected acetyl-coenzyme A to increase cell intrinsic steroidogenesis. The enhanced steroidogenesis in Bat3-deficient DC suppressed T cell response in a paracrine manner. Our findings identified Bat3 as an endogenous regulator of DC function, which has implications for DC-based immunotherapies.

Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.

To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.

TIM-3 restrains anti-tumour immunity by regulating inflammasome activation.

T cell immunoglobulin and mucin-containing molecule 3 (TIM-3), first identified as a molecule expressed on interferon-γ producing T cells1, is emerging as an important immune-checkpoint molecule, with therapeutic blockade of TIM-3 being investigated in multiple human malignancies. Expression of TIM-3 on CD8+ T cells in the tumour microenvironment is considered a cardinal sign of T cell dysfunction; however, TIM-3 is also expressed on several other types of immune cell, confounding interpretation of results following blockade using anti-TIM-3 monoclonal antibodies. Here, using conditional knockouts of TIM-3 together with single-cell RNA sequencing, we demonstrate the singular importance of TIM-3 on dendritic cells (DCs), whereby loss of TIM-3 on DCs-but not on CD4+ or CD8+ T cells-promotes strong anti-tumour immunity. Loss of TIM-3 prevented DCs from expressing a regulatory program and facilitated the maintenance of CD8+ effector and stem-like T cells. Conditional deletion of TIM-3 in DCs led to increased accumulation of reactive oxygen species resulting in NLRP3 inflammasome activation. Inhibition of inflammasome activation, or downstream effector cytokines interleukin-1ß (IL-1ß) and IL-18, completely abrogated the protective anti-tumour immunity observed with TIM-3 deletion in DCs. Together, our findings reveal an important role for TIM-3 in regulating DC function and underscore the potential of TIM-3 blockade in promoting anti-tumour immunity by regulating inflammasome activation.

Tim-3: A co-receptor with diverse roles in T cell exhaustion and tolerance.

T cell inhibitory co-receptors play a crucial role in maintaining the balance between physiologic immune responses and maladaptive ones. T cell immunoglobulin and mucin domain-containing-3 (Tim-3) is a unique inhibitory co-receptor in that its expression is chiefly restricted to interferon (IFN)γ-producing CD4+ and CD8+ T cells. Early reports firmly established its importance in maintaining peripheral tolerance in transplantation and autoimmunity. However, it has become increasingly clear that Tim-3 expression on T cells, together with other check-point molecules, in chronic infections and cancers can hinder productive immune responses. In this review, we outline what is currently known about the regulation of Tim-3 expression, its ligands and signaling. We discuss both its salutary and deleterious function in immune disorders, as well as the T cell-extrinsic and -intrinsic factors that regulate its function.

Different clinical outcomes in Crohn's disease patients with esophagogastroduodenal, jejunal, and proximal ileal disease involvement: is L4 truly a single phenotype?

BACKGROUND: The Montreal classification defines L4 Crohn's disease (CD) as any disease location proximal to the terminal ileum, which anatomically includes L4-esophagogastroduodenal (EGD), L4-jejunal, and L4-proximal ileal involvement. L4-jejunal disease was established to be associated with poor prognosis. However, the outcome of patients with L4-proximal ileal disease or L4-EGD remains to be clarified. Our study aimed to investigate whether the outcome differs among CD patients with L4-EGD, L4-jejunal, and L4-proximal ileal disease. METHODS: In our retrospective cohort study, 483 patients with confirmed CD were included. The primary outcome was intestinal surgery. Demographic features and outcomes were compared among L4-EGD, L4-jejunal, and L4-proximal ileal disease. RESULTS: Thirty-nine (8.1%) patients had isolated L4 disease, whereas 146 patients had L4 as well as concomitant L1, L2, or L3 disease. During a median follow up of 5.8 years, L4 patients were more likely to have intestinal surgeries compared to non-L4 patients (31% versus 16%, p < 0.001). The percentage of L4-jejunal patients who underwent surgery was higher than that of L4-proximal ileal (66% versus 28%, p < 0.001), and both of these subtypes of L4 were at higher risk for intestinal resection compared to L4-EGD patients (66% and 28% versus 9%, respectively, p < 0.001 and p < 0.05). On multi-variable analysis, L4-jejunal (HR 3.08; 95% CI 1.30-7.31) and L4-proximal ileal disease (HR 1.83; 95% CI 1.07-3.15) were independent predictors for intestinal resection. CONCLUSIONS: L4 disease had worse prognosis compared to non-L4 disease. Within L4 disease, phenotype of L4-jejunal and L4-proximal ileal disease indicated higher risk for intestinal surgery. It might be justified to further characterize the L4 phenotype of the Montreal classification into three specific subgroups including L4-EGD, L4-jejunal, and L4-proximal ileal disease, similar to the Paris classification of pediatric patients.

[Clinical manifestations of Crohn's disease misdiagnosed as appendicitis].

OBJECTIVE: To analyze the clinical manifestations and identify independent diagnostic predictive factors for Crohn's disease (CD) initially diagnosed as appenicitis and treated by surgery. METHODS: The medical records of patients diagnosed as acute appendicitis upon admission and treated by surgical operation in the First Affiliated Hospital of Sun Yat-sen University from January 2000 to December 2014 were retrospectively analyzed. Based on postoperative pathological examination and clinical examination results, 28 CD patients were identified (CD group), and for each CD case, 3 controls with confirmed diagnosis of appendicitis were included (appendicitis group, n=84). Clinical manifestations and laboratory examination results of the two groups were analyzed with multivariable Logistic regression to determine independent diagnostic predictive factors for CD initially misdiagnosed as appendicitis. RESULTS: Altogether 112 patients were enrolled, with a male-to-female ratio of 1.04:1 (57:55), and a median age of 36 years. No significant differences were found in gender, age, and body temperature between the CD group and appendicitis group (all P>0.05). In the appendicitis and CD groups, median duration (Q1-Q3) of abdominal pain was 24 (14-48) hours vs 216 (96-384) hours, proportion of patients with lower right abdominal pain was 98.8% (83/84) vs 75.0% (21/28), proportion of patients with shifting lower right abdominal pain was 98.8% (83/84) vs 7.1% (2/28), proportion of patients with local lower right peritonitis was 95.2% (80/84) vs 53.6% (15/28), proportion of patients with change of bowel emptying habit or stool consistency was 7.1% (6/84) vs 46.4% (13/28), proportion of patients with history of chronic abdominal pain or diarrhea was 10.7% (9/84) vs 75.0% (21/28), preoperative white blood cell count was (14.08±4.13)×10(9)/L vs (8.00±3.42)×10(9)/L, preoperative neutrophil count was (11.34±4.10)×10(9)/L vs (5.58±3.22)×10(9)/L, preoperative hemoglobin was (139.52±19.90) g/L vs (107.65±21.68) g/L, preoperative red blood cell count was (4.85±0.74)×10(12)/L vs (4.28±0.87)×10(12)/L, and preoperative platelet count was (220.68±74.47)×10(9)/L vs (302.09±71.65)×10(9)/L, all with significant differences (all P<0.05). Multivariable analysis showed that change of bowel emptying habit and stool consistency (OR=36.712, 95%CI: 1.672-806.103, P=0.022), medical history of chronic abdominal pain or diarrhea (OR=60.142, 95%CI: 4.501-803.573, P=0.002), lower preoperative hemoglobin level (OR=0.909, 95%CI: 0.858-0.963, P=0.001), and higher platelet count (OR=1.027, 95%CI: 1.007-1.047, P=0.008) were independent predictive factors for CD. CONCLUSIONS: CD should be considered in cases initially diagnosed as appendicitis with change of bowel emptying habit and stool consistency, medical history of chronic abdominal pain or diarrhea, anemia, and increased platelet count.

[Clinical characteristics and risk factors of refractory Crohn's disease].

OBJECTIVE: To analyze the clinical characteristics and risk factors of refractory Crohn's disease (CD). METHODS: All clinical data of confirmed consecutive CD patients were collected from our hospital between January 2003 and June 2013. The patients' demographic data, clinical features, therapeutic regimens and laboratory examinations were analyzed. A multivariate Logistic regression was performed to identify the risk factors of refractory CD. RESULTS: (1) A total of 402 confirmed CD patients were recruited for analysis. The prevalence of refractory CD was 33.8% (136/402). The rates of steroid-dependency was 37.0% (97/262) in 262 patients with a history of steroid use and the rate of thiopurines ineffectiveness was 26.9% (79/294) in 294 patients with a history of thiopurines-use; (2) Univariate analysis showed that disease location (L3 type), abdominal pain, diarrhea, fever, abdominal tenderness, perianal lesion, steroid use, AZA/6-MP use, leucocyte, hemoglobin (Hb), platelet level and high-sensitivity C-reactive protein (HsCRP) were significantly different between refractory and non-refractory CD patients (all P < 0.05) . Multivariate Logistic regression showed that steroid use (OR = 6.516, 95% CI: 2.884-14.722, P = 0.000) and low Hb (OR = 1.023, 95% CI: 1.008-1.037, P = 0.002) were independent risk factors related to refractory CD; (3) Univariate analysis showed that Hb level, erythrocyte sedimentation rate (ESR) were significantly different between steroid-dependent and non-steroid-dependent groups (all P < 0.05) . Multivariate Logistic regression showed that only low Hb level (OR = 1.021, 95% CI: 1.006-1.036, P = 0.005) was an independent risk factor related to steroid-dependency; (4) Univariate analysis showed that disease location (L3 type), perianal lesion, abdominal pain, diarrhea, fever, abdominal tenderness, platelet level, steroid use, steroid-dependency were significantly different between thiopurines-ineffective and thiopurines-effective groups (all P < 0.05) . Multivariate Logistic regression showed that perianal lesion (OR = 2.085, 95% CI: 1.007-4.039, P = 0.029), abdominal tenderness (OR = 2.943, 95% CI: 1.452-5.964, P = 0.003) and steroid-dependency (OR = 3.599, 95% CI: 1.847-7.013, P = 0.000) were independent risk factors related to thiopurines-ineffectiveness. CONCLUSIONS: Nearly one third CD patients became refractory during the course of disease. Low Hb and steroid use are independent risk factors. Low Hb is an independent risk factor related to steroid-dependency. Perianal disease, abdominal tenderness and steroid-dependency are independent risk factors related to thiopurines- ineffectiveness.

Opposite effects of interferon regulatory factor 1 and osteopontin on the apoptosis of epithelial cells induced by TNF-α in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by a damaged intestinal epithelium barrier. Interferon regulatory factor 1 (IRF1) and osteopontin (OPN) regulate cell survival and growth in a variety of circumstances but their effects on the intestinal epithelium have not been elucidated. In this study, we sought to determine the effects of OPN on intestinal epithelial cells under conditions of tumor necrosis factor (TNF)-α-induced inflammation and whether IRF1 regulates OPN expression, the activation of downstream pathways, and inflammatory responses. METHODS: The expression levels of OPN and IRF1 were assessed by immunohistochemical analyses of human IBD and experimental mouse colitis. The effects of IRF1 and OPN on inflammatory responses were investigated in vitro in NCM460 and Caco-2 cells stimulated by TNF-α. Changes in p-AKT, p-P38, and p-ERK levels were quantified by western blotting assays. The regulation of OPN expression by IRF1 was determined by luciferase activity and chromatin immunoprecipitation assays. RESULTS: IRF1 was upregulated in human IBD and in the colon epithelium of mice with dextran sulfate sodium-induced colitis. Additionally, IRF1 was correlated with high-sensitivity C-reactive protein, erythrocyte sedimentation rate, Crohn's disease activity index, Crohn's disease endoscopic index of severity, and simple endoscopic score for Crohn's disease in Crohn's disease and with high-sensitivity C-reactive protein, erythrocyte sedimentation rate, Mayo score, Baron score, modified Baron score, Rachmilewitz score, ulcerative colitis endoscopic index of severity, ulcerative colitis colonoscopic index of severity, and disease duration in ulcerative colitis. The expression of OPN was significantly decreased in patients with IBD compared with controls and in dextran sulfate sodium-induced experimental colitis and was also inversely correlated with clinical and endoscopic activities in both Crohn's disease and ulcerative colitis. TNF-α treatment upregulated IRF1 and diminished OPN in both NCM460 and Caco-2 cells. The overexpression of OPN and rhOPN ameliorated the apoptosis induced by TNF-α, whereas the overexpression of IRF1 aggravated apoptosis, indicating opposite effects of OPN and IRF1 in inflamed epithelial cells. The luciferase and chromatin immunoprecipitation assays showed that IRF1 transcriptionally modulated the expression of OPN. TNF-α inhibited the OPN-induced upregulation of p-ERK, p-P38, and p-AKT. CONCLUSIONS: Our data suggest that during intestinal inflammation, the TNF-α-mediated activation of IRF1 is related to the subsequent suppression of OPN expression, further reducing p-AKT, p-P38, and p-ERK activities and resulting in aggravation of the injury to intestinal epithelial cells.

[The efficacy and safety of methotrexate in refractory Crohn's disease].

OBJECTIVE: To assess the efficacy and safety of methotrexate(MTX) on refractory Crohn's disease(CD). METHODS: A total of 35 consecutive refractory CD patients in the First Affiliated Hospital of Sun Yat-Sen University treated with MTX were retrospectively analyzed. Clinical data from June 2004 to December 2012 were collected from the database of inflammatory bowel disease (IBD) center. Clinical responses and drug side effects were recorded and analyzed. RESULTS: Thirty-five refractory CD patients were identified including 23 cases intolerant to azathioprine (AZA)/6-mercaptopurine(6-MP), six cases ineffective to AZA/6-MP, 19 cases dependent on steroid. After treatment of MTX for 12 weeks [15(5-20) mg/week], a clinical response was obtained in 80% patients (28/35), including 51.4% (18/35) in remission and 28.6% (10/35) in improvement. The median Crohn's disease activity index (CDAI) scores at the onset and 12 weeks after MTX therapy were 99.2 (75.8, 174.7) and 61.5 (36.0, 106.6) respectively. The median single dose and duration of MTX were 15 (5-20) mg/week and 6.0(0.5-53.0) months respectively. The median cumulative dose was 480 (20-2615) mg. Among the 26 patients dependent on steroid, 21 achieved discontinuation of steroid with a median time of 10 (6-20) weeks after treatment of MTX. Side effects were recorded in 12 patients (34.3%), but usually mild and improved after drug with drawal. CONCLUSIONS: MTX is an effective and steroid-sparing agent for refractory CD. Side effects of MTX are mild and tolerable.

[The prevalence and characteristics of anemia in Crohn's disease in the Chinese].

OBJECTIVE: To investigate the prevalence and characteristics of anemia among patients with Crohn's disease (CD) in Chinese population and identify the possible risk factors. METHODS: A cross-sectional study was performed in 441 patients with CD enrolled from the First Affiliated Hospital of Sun Yat-Sen University between January 2003 and May 2012. The prevalence, severity, type of anemia in these patients was assessed when diagnosis was confirmed. A multivariate logistic regression including 122 patients was performed to screen risk factors of anemia. RESULT: The prevalence of anemia was 64.4% (284/441) with 69.0% (196/284) mild anemia, 28.9% (82/284) moderate anemia and 2.1% (6/284) severe anemia. The most common morphological classification was hypochromic microcytic anemia (43.7%, 124/284). Multivariate logistic regression showed the predictive factors for anemia were higher levels of modified Crohn's disease activity index (CDAI) (OR = 1.007, 95% CI 1.002-1.013), platelet count (OR = 1.007, 95% CI 1.001-1.012), erythrocyte sedimentation rate (OR = 1.024, 95% CI 1.000-1.048), penetrating behavior (OR = 16.952, 95% CI 2.626-108.626), structuring behavior (OR = 6.717, 95% CI 1.583-28.507), older age at diagnosis (OR = 1.065, 95% CI 1.012-1.121),and lower body mass index (BMI) (OR = 0.769, 95% CI 0.633-0.935). CONCLUSIONS: Anemia is a common complication in patients with CD among Chinese population. Activity of the underlying disease, older age at diagnosis, penetrating or structuring disease behavior and low BMI are the risk factors.