CTLA4-Ig prevents development of neutralizing antibody formation after continuous treatment with human FVIII in HA rats.

INTRODUCTION: Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models. AIM: To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment. METHODS: Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study. RESULTS: The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved. CONCLUSION: In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.

Cesarean section increases sensitivity to oxazolone-induced colitis in C57BL/6 mice.

Children born by cesarean section (CS) have an increased risk of developing inflammatory bowel disease (IBD), possibly due to skewed microbial colonization during birth and consequently impaired bacterial stimulation of the developing immune system. The aim of this study was to investigate the association between CS and experimental colitis in a murine model of IBD. It was hypothesized that CS aggravates colonic inflammation due to a change in gut microbiota (GM) composition. C57BL/6 mice, delivered by CS or vaginal delivery (VD), were intra-rectally challenged with oxazolone at 8 weeks of age and monitored for colitis symptoms. The results showed that CS delivered mice experienced an increased body weight loss and colon weight, together with higher colonic concentrations of TNF-α and MPO compared with VD mice. Increased infiltration of inflammatory cells was present in CS delivered mice, as well as a downregulation in expression of the gut integrity genes occludin and tight junction protein 1 indicative of an impaired barrier function. The GM from CS delivered mice without colitis partly contributed to the increase in colitis symptoms when inoculated into germ-free recipient mice. In conclusion, CS increased sensitivity to oxazolone induced colitis in mice.

Cesarean Section Induces Microbiota-Regulated Immune Disturbances in C57BL/6 Mice.

Epidemiological studies have shown that children born by cesarean section (CS) are at higher risk of developing chronic inflammatory diseases, and it has been suggested that a skewed gut microbial colonization process early in life and altered priming of the immune system are causative. The aim of this study was to clarify whether impaired regulatory immunity in CS-delivered C57BL/6 mice is dependent on gut microbiota (GM) disturbances. The GM of conventionally bred mice born by CS differed clearly from mice born by vaginal delivery. The proportion of regulatory T cells was reduced in mice born by CS, whereas the invariant NKT (iNKT) cell subset was increased compared with vaginal delivery mice. In addition, regulatory markers (Foxp3, Il10, Ctla4) and macrophage markers (Cd11c, Egr2, Nos2) were downregulated, whereas iNKT markers (Il4, Il15) were upregulated in ileum of CS-delivered mice. The GM of CS-delivered mice was sufficient to transfer the shifts in immunity associated with delivery mode when inoculated into germ-free mice. Feeding a prebiotic diet reestablished gene expression of intestinal immune markers and iNKT cells in CS mice but was not sufficient to restore the level of regulatory T cells. The results support that CS delivery is associated with microbiota-mediated shifts in regulatory immunity and, therefore, provide a basis for future microbiota-directed therapeutics to infants born by CS.

Evaluating IL-21 as a Potential Therapeutic Target in Crohn's Disease.

BACKGROUND AND AIM: Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohn's Disease (CD) and could be a potential new therapeutic target in CD. METHODS: In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R-/- T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. RESULTS: In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2-/- mice receiving CD4+CD45RBhighIL-21R-/- T cells developed less severe colitis compared to Rag2-/- mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. CONCLUSION: Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.

Characterization of Growth Hormone Resistance in Experimental and Ulcerative Colitis.

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH-insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.

High-resolution gene expression profiling using RNA sequencing in patients with inflammatory bowel disease and in mouse models of colitis.

BACKGROUND AND AIMS: Proper interpretation of data from preclinical animal studies requires thorough knowledge of the pathophysiology of both the human disease and animal models. In this study, the expression of inflammatory bowel disease [IBD]-associated genes was characterised in mouse models of colitis to examine the underlying molecular pathways and assess the similarity between the experimental models and human disease. METHODS: RNA sequencing was performed on colon biopsies from Crohn's disease [CD] patients, ulcerative colitis [UC] patients and non-IBD controls. Genes shown to be significantly dysregulated in human IBD were used to study gene expression in colons from a piroxicam-accelerated colitis interleukin-10 knockout [PAC IL-10 k.o.], an adoptive transfer [AdTr] and a dextran sulfate sodium [DSS] colitis mouse model. RESULTS: Of 115 literature-defined genes linked to IBD, 92 were significantly differentially expressed in inflamed mucosa of CD and/or UC patients compared with non-IBD controls. The most upregulated genes were shared by both diseases, including REG1A, LCN2, NOS2, CXCL1-2, and S100A9. Of those 92 IBD-associated genes, 71 [77%] were significantly dysregulated in PAC IL-10 k.o. mice, whereas 59 [64%] were significantly dysregulated in AdTr mice compared with wild-type controls. Some of the most upregulated genes, including S100a8-9, Nos2, and Lcn2, were shared by the colitis models and correlated with disease activity. CONCLUSIONS: IBD and experimental murine colitis have a high degree of similarity in the colonic transcriptional profile, probably secondary to non-specific inflammatory processes. However, differences do exist between models, emphasising the need for careful selection and interpretation of qualified animal models in preclinical research.

IL-33 promotes GATA-3 polarization of gut-derived T cells in experimental and ulcerative colitis.

BACKGROUND: In the respiratory mucosa, interleukin (IL)-33, has been shown to enhance T helper 2 (TH2)-type responses through the master regulatory gene GATA-3. IL-33 is upregulated in ulcerative colitis (UC), and the aim was to assess if IL-33 holds a similar key position in the shaping of the immune response in experimental colitis (piroxicam-accelerated colitis (PAC) in IL-10 (-/-) mice, dextran sodium sulfate (DSS) model) and UC. METHODS: Colonic IL-33 expression was determined in UC (8 active UC, 8 quiescent UC, and 7 controls) and experimental colitis. Mesenteric lymph node (MesLN) T cells were isolated from PAC IL-10 (-/-) mice and stimulated with IL-33. RESULTS: The colonic IL-33 expression was significantly upregulated all forms of colitis (P < 0.01) and correlated with disease severity score and inflammation (P < 0.001), and with GATA-3 expression levels (P < 0.01); no correlation with the TH1-specific T-bet expression was observed. MesLN T cells stimulated with IL-33 had increased GATA-3 expression, and showed an IL-33 dose-dependent increase in secreted TH2-type cytokines, whereas this effect was abolished by blocking IL-33 signaling. The non-TH2-type cytokine IL-17 was upregulated by IL-33 but in a T cell receptor dependent manner, as opposed to TH2-type cytokines, which required only IL-33 stimulation. CONCLUSIONS: The study demonstrates that intestinal IL-33 is capable of inducing GATA-3 in mucosal T cells, and suggests that IL-33 is a key mediator of pathological TH2 and non-TH2-type responses in intestinal inflammation. Blocking IL-33 signaling could be a feasible option in the treatment of UC.

Predictive validity and immune cell involvement in the pathogenesis of piroxicam-accelerated colitis in interleukin-10 knockout mice.

Piroxicam administration is a method for induction of enterocolitis in interleukin-10 knockout (IL-10 k.o.) mice. The piroxicam-accelerated colitis (PAC) IL-10 k.o. model combines a dysregulated immune response against the gut microbiota with a decreased mucosal integrity. The predictive validity and pathogenic mechanisms of the model have not been thoroughly investigated. In this study, IL-10 k.o. mice received piroxicam in the chow, and model qualification was performed by examining the efficacy of prophylactic anti-IL-12/23p40 monoclonal antibody (mAb), anti-TNFα mAb, cyclosporine A (CsA) and oral prednisolone treatment. To evaluate cell involvement in the disease pathogenesis, specific cell subsets were depleted by treatment with anti-CD4 mAb, anti-CD8 mAb or clodronate-encapsulated liposomes. T cell receptor co-stimulation was blocked by CTLA4-Ig. Cytokine profiling ELISAs and calprotectin immunohistochemistry were performed on colon tissue. Treatments with anti-IL-12/23p40 mAb and CsA prevented disease in PAC IL-10 k.o. mice and reduced IFNγ, IL-17A, MPO and calprotectin levels in colon. Anti-TNFα mAb treatment caused amelioration of selected clinical parameters. No effect of prednisolone was detected. Depletion of CD8(+) cells tended to increase mortality, whereas treatment with anti-CD4 mAb or CTLA4-Ig had no significant effect on disease development. Clodronate liposome treatment induced a loss of body weight; nevertheless macrophage depletion was associated with a significant reduction in colonic pathology. In conclusion, reference drugs with known efficacy in severe inflammatory bowel disease were efficacious in the PAC IL-10 k.o. model. Our data indicate that in this model macrophages are a main driver of colitis, whereas CD4(+) cells are not.

Characterisation of enterocolitis in the piroxicam-accelerated interleukin-10 knock out mouse--a model mimicking inflammatory bowel disease.

BACKGROUND: In inflammatory bowel disease a defective mucosal barrier, a dysregulated immune response and an excessive reactivity against the gut microbiota are assumed to cause a breakdown of the intestinal homeostasis and lead to chronic inflammation. Piroxicam treatment is a method for induction of colitis in IL-10 k.o. mice, which integrates a dysfunction of both the intestinal barrier and the immune system. However, the translational value of this model has not been thoroughly clarified. AIM: To characterise the piroxicam-accelerated colitis (PAC) IL-10 k.o. model with respect to clinical features, pathogenic mechanisms and its ability to respond to existing therapies. METHODS: The PAC IL-10k.o. model was established on a C57BL/6J background and the clinical manifestations, immunological mechanisms and efficacy of ampicillin and anti-IL-12/23p40 treatment were assessed. RESULTS: The PAC IL-10 k.o. mice developed weight loss and diarrhoea, and colonoscopy revealed a thickened granulomatous mucosa. Histological evaluation of ileum and colon showed Crohn's disease-like changes with pronounced hyperplasia and focal transmural inflammation. Ileitis was also observed in piroxicam treated wild type mice. The total number of neutrophils, monocytes and natural killer cells was elevated in the blood compared to IL-10 k.o. and wild type mice, indicating a role of the innate immune system in the pathogenesis. These findings were supported by analyses of the intestinal cytokine profile. Ampicillin and anti-IL-12/23p40 treatment significantly suppressed disease in the model. CONCLUSION: The PAC IL-10 k.o. model resembles several features of Crohn's disease and could be a useful in vivo model in preclinical research.

TNF-α-induced down-regulation of CDX2 suppresses MEP1A expression in colitis.

BACKGROUND/AIMS: High levels of pro-inflammatory cytokines are linked to inflammatory bowel disease (IBD). The transcription factor Caudal-related homeobox transcription factor 2 (CDX2) plays a crucial role in differentiation of intestinal epithelium and regulates IBD-susceptibility genes, including meprin 1A (MEP1A). The aim was to investigate the expression of CDX2 and MEP1A in colitis; to assess if they are regulated by tumor necrosis factor-α (TNF-α), and finally to reveal if CDX2 is involved in a TNF-α-induced down-regulation of MEP1A. METHODS: Expression of CDX2 and MEP1A was investigated in colonic biopsies of ulcerative colitis (UC) patients and in dextran sodium sulfate (DSS)-induced colitis. CDX2 protein expression was investigated by immunoblotting and immunohistochemical procedures. CDX2 and MEP1A regulation was examined in TNF-α-treated Caco-2 cells by reverse transcription-polymerase chain reaction and with reporter gene assays, and the effect of anti-TNF-α treatment was assessed using infliximab. Finally, in vivo CDX2-DNA interactions were investigated by chromatin immunoprecipitation. RESULTS: The CDX2 and MEP1A mRNA expression was significantly decreased in active UC patients and in DSS-colitis. Colonic biopsy specimens from active UC showed markedly decreased CDX2 staining. TNF-α treatment diminished the CDX2 and MEP1A mRNA levels, a decrease which, was counteracted by infliximab treatment. Reporter gene assays showed significantly reduced CDX2 and MEP1A activity upon TNF-α stimulation. Finally, TNF-α impaired the ability of CDX2 to interact and activate its own, as well as the MEP1A expression. CONCLUSIONS: The present results indicate that a TNF-α-mediated down-regulation of CDX2 can be related to suppressed expression of MEP1A during intestinal inflammation.

Confirmation of a disease model of pemphigus vulgaris: characterization and correlation between disease parameters in 90 mice.

Pemphigus vulgaris (PV) is a chronic autoimmune bullous disease associated with immunoglobulin G (IgG) autoantibodies against desmoglein 3 (Dsg3). Previously, a mouse model of PV was established by adoptive transfer of naive splenocytes from Dsg3(-/-) mice to Rag2(-/-) mice. The model is unique as Dsg3-specific naive lymphocytes from Dsg3(-/-) mice can be primed and activated by the endogenous Dsg3 in recipient mice, resulting in pathogenic anti-Dsg3 IgG without any active immunization. Here, we show that PV occurs after both intravenous (i.v.) and intraperitoneal (i.p.) transfer of naive splenocytes. We evaluated the robustness of the model by comparing engraftment as well as PV phenotype using several disease parameters. While engraftment of spleen cells was significantly better after i.p. transfer, anti-Dsg3 IgG antibody production, IgG deposition and disease score were comparable after both i.v. and i.p. cell transfer. Thus, transferred cells can be primed, activated and gain effector function. However, we detected heterogeneity in disease development, as only 46% of the mice developed hair loss, whereas 76% of the mice developed anti-Dsg3 IgG. We also tested cyclophosphamide in the model, as this drug is reported to be beneficial to PV patients. Cyclophosphamide significantly inhibited disease development in a preventive setting, and mice were free of symptoms 35 days after discontinuing the treatment. We have successfully confirmed the induction of PV after both i.v. and i.p. transfer. In addition, we have shown that this model can be used for evaluation of immunosuppressive drugs.

In vivo control of diabetogenic T-cells by regulatory CD4+CD25+ T-cells expressing Foxp3.

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.

CD4+CD25+ regulatory T cells: I. Phenotype and physiology.

The immune system protects us against foreign pathogens. However, if fine discrimination between self and non-self is not carried out properly, immunological attacks against self may be launched leading to autoimmune diseases, estimated to afflict up to 5% of the population. During the last decade it has become increasingly clear that regulatory CD4+CD25+ T cells (Treg cells) play an important role in the maintenance of immunological self-tolerance, and that this cell subset exerts its function by suppressing the proliferation or function of autoreactive T cells. Based on human and murine observations, this review presents a characterization of the phenotype and functions of the Treg cells in vitro and in vivo. An overview of the surface molecules associated with and the cytokines produced by the Treg cells is given and the origin, activation requirements and mode of action of the Treg cells are discussed. Finally, we address the possibility that Treg cells may play a central role in immune homeostasis, regulating not only autoimmune responses, but also immune responses toward foreign antigens.

CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects.

Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4+CD25+ T cells (Treg cells) have been shown to play an important role in the maintenance of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of Treg cells. The present overview deals with the thymic origin of Treg cells and their role in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of Treg cells.