Reversible changes in serum immunoglobulin galactosylation during the immune response and treatment of inflammatory autoimmune arthritis.

OBJECTIVES: Improved DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology was used to monitor the changes in the galactosylation status of serum immunoglobulins during the immune response and therapy of autoimmune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in susceptible DBA/1 mice and the undergalactosylation status (UGS) of serum immunoglobulins was determined using the improved DSA-FACE technology. Prophylactic intravenous tolerisation with type II collagen as well as semitherapeutic treatment with dexamethasone (DEX) were performed and UGS was analysed. Next, the serum immunoglobulin glycosylation profiles of patients with rheumatoid arthritis (RA) and spondyloarthropathy (SpA) were studied and changes in the UGS scores during anti-tumour necrosis factor (TNF)alpha therapy followed. RESULTS: In the longitudinal CIA study, the undergalactosylation state of immunoglobulins was found to be significantly correlated with the clinical arthritis scores. Upon collagen-specific tolerisation as well as glucocorticoid semitherapeutic treatment, improvement of the clinical arthritis scores correlated with decreased levels of UGS. It was also demonstrated that withdrawal of DEX was associated with an increased UGS score. Interestingly, reversibility in the UGS was also shown during treatment of patients with RA and SpA with anti-TNFalpha. CONCLUSIONS: These findings demonstrate that the UGS of serum immunoglobulins changes during the disease course of CIA and that this UGS is inhibited by antigen-specific and antigen-independent treatment procedures. The observation that Ig galactosylation is a reversible process is also documented during treatment of patients with RA and SpA with anti-TNFalpha.

Genetically engineered Lactococcus lactis secreting murine IL-10 modulates the functions of bone marrow-derived dendritic cells in the presence of LPS.

Oral delivery of IL-10 by genetically modified Lactococcus lactis (LL-pTmIL10) has been shown to efficiently reduce intestinal inflammation in mice with chronic colitis, but the mechanisms involved have not been elucidated. It has been suggested that IL-10 controls intestinal inflammation by inhibiting microbe-induced activation of dendritic cells. We therefore investigated whether LL-pTmIL10 can modulate the functions of bone marrow-derived dendritic cells (BM-DC) responding to LPS. Incubation of these cells with LL-pTmIL10 or with the control strain LL-pTREX reduced their ability to activate allogeneic T-cell proliferation. However, in contrast to LL-pTREX, LL-pTmIL10 inhibited the LPS-stimulated secretion of MCP-1 by BM-DC and reduced the synergistic up-regulation of IL-12/IL-23p40. In addition, LL-pTmIL10 treatment of LPS-stimulated BM-DC significantly inhibited their capacity to induce strong secretion of IL-17 by CD4+ T cells. Our data suggest that the beneficial effects of LL-pTmIL10 treatment during chronic colitis might involve inhibition of CD4+ Th17 cells and a reduced accumulation of these cells as well as other immune cells at the site of inflammation.

Inflamed intestinal mucosa features a specific epithelial expression pattern of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic ß-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Tumoral environment triggers transcript anomalies in established tumors: induction of altered gene expression and of aberrant, truncated and B2 repeat-containing gene transcripts.

In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR)-based subtraction suppression hybridization (SSH) to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.

Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

Murine trefoil factor 3 does not directly modulate LPS-mediated dendritic cell function.

Peptides of the trefoil factor family (TFF) are expressed along the gastro-intestinal tract. They protect mucous epithelia from damage and contribute to mucosal repair, which is essential for preventing inflammation. Moreover, it has been suggested that TFF2 and TFF3, in particular, play a role in regulating immune responses. Depending on their activation status, dendritic cells (DC) can initiate either tolerance or immunity. This study, by comparing LPS-induced maturation of mTFF3-treated DC and non-treated DC, investigated whether murine TFF3 directly regulated DC function. mTFF3-treated DC and non-treated DC did not differ phenotypically or functionally. Both populations expressed, both before and after LPS-stimulation, similar levels of co-stimulatory molecules and cytokines, and were both efficient stimulators of T-cells. Our results suggest that mTFF3 does not govern immune responses on the level of DC function.

CARD15 variants determine a disturbed early response of monocytes to adherent-invasive Escherichia coli strain LF82 in Crohn's disease.

Caspase activation and recruitment domain 15 (CARD15) and Toll-like receptor 4 (TLR4) are respectively intracellular and membrane-bound receptors for bacterial cell wall components [respectively muramyl dipeptide (MDP) and lipopolysaccharide (LPS)]. Polymorphisms in CARD15 and TLR4 have been linked with Crohn's disease (CD). Adherent-invasive Escherichia coli (AIEC) strains with particular adhesion and invasion characteristics have been specifically associated with CD ileal mucosa. The aim of this study was to investigate the functional impact of these polymorphisms on monocytes in patients with CD, in response to MDP, LPS and AIEC strain LF82. Monocytes were isolated from 40 patients with CD using magnetic cell sorting, stimulated with LPS or MDP or infected with AIEC. IL-1beta, IL-6, IL-8, IL-10, IL-12 and tumour necrosis factor alpha induction was assessed using quantitative real time-polymerase chain reaction, Cytometric Bead Array and ELISA. Bacterial intracellular survival and replication was assessed using a gentamicin protection assay. Results were linked with the presence of CARD15 and TLR4 polymorphisms. Monocytes of patients with CARD15 polymorphisms showed an early reduced cytokine response (IL-1beta, IL-6 and IL-10) to infection with AIEC, which was restored after 20 h. A gene-dose effect was seen, comparing wild-types, heterozygotes and homozygotes. We found no differences in intracellular survival and replication of AIEC. Heterozygous carriage of TLR4 polymorphisms did not influence monocyte response. In conclusion, patients with CD carrying CARD15 polymorphisms show a disturbed early inflammatory monocyte response after infection with AIEC strain LF82. For the first time, a functional defect was detected in single heterozygous carriers. These findings reflect the potential role of a genetically altered host response to disease-related bacteria in the pathogenesis of CD.

Altered gut transcriptome in spondyloarthropathy.

BACKGROUND: Intestinal inflammation is a common feature of spondyloarthropathy (SpA) and Crohn's disease. Inflammation is manifested clinically in Crohn's disease and subclinically in SpA. However, a fraction of patients with SpA develops overt Crohn's disease. AIMS: To investigate whether subclinical gut lesions in patients with SpA are associated with transcriptome changes comparable to those seen in Crohn's disease and to examine global gene expression in non-inflamed colon biopsy specimens and screen patients for differentially expressed genes. METHODS: Macroarray analysis was used as an initial genomewide screen for selecting a comprehensive set of genes relevant to Crohn's disease and SpA. This led to the identification of 2625 expressed sequence tags that are differentially expressed in the colon of patients with Crohn's disease or SpA. These clones, with appropriate controls (6779 in total), were used to construct a glass-based microarray, which was then used to analyse colon biopsy specimens from 15 patients with SpA, 11 patients with Crohn's disease and 10 controls. RESULTS: 95 genes were identified as differentially expressed in patients with SpA having a history of subclinical chronic gut inflammation and also in patients with Crohn's disease. Principal component analysis of this filtered set of genes successfully distinguished colon biopsy specimens from the three groups studied. Patients with SpA having subclinical chronic gut inflammation cluster together and are more related to those with Crohn's disease. CONCLUSION: The transcriptome in the intestine of patients with SpA differs from that of controls. Moreover, these gene changes are comparable to those seen in patients with Crohn's disease, confirming initial clinical observations. On the basis of these findings, new (genetic) markers for detection of early Crohn's disease in patients with SpA can be considered.

CARD15 gene polymorphisms in patients with spondyloarthropathies identify a specific phenotype previously related to Crohn's disease.

BACKGROUND: The association between spondyloarthropathy and Crohn's disease is well known. A risk for evolution to Crohn's disease has already been shown in the subgroup of patients with spondyloarthropathy associated with chronic gut inflammation. OBJECTIVE: To investigate whether the reported polymorphisms in the CARD15 gene, a susceptibility gene for Crohn's disease, are associated with the presence of preclinical intestinal inflammation observed in spondyloarthropathies. METHODS: 104 patients with spondyloarthropathies were studied. All underwent ileocolonoscopy with biopsies between 1983 and 2004. The prevalence of three single nucleotide polymorphisms in the CARD15 gene (R702W, G908R, and 1007fs) was assessed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR); the patients were compared with an ethnically matched Crohn's disease population and a control population. RESULTS: The carrier frequency of R702W, G908R, or 1007fs variants in the spondyloarthropathy populations (20%) was similar to the control population (17%), but increased to 38% in the spondyloarthropathy subgroup with chronic gut inflammation. This frequency was significantly higher than in the other spondyloarthropathy subgroups (p = 0.001) or the control group (p = 0.006), but not different from the Crohn's disease group (49%) (NS). This indicates that CARD15 polymorphisms are associated with a higher risk for development of chronic gut inflammation. CONCLUSIONS: CARD15 gene polymorphisms clearly identify a subgroup of patients with spondyloarthropathies associated with chronic intestinal inflammation.

Macrophages induce cellular immunity by activating Th1 cell responses and suppressing Th2 cell responses.

Differentiation of naive CD4+ T cells (Th0) into Th1 or Th2 cells determines whether antigen will raise a cellular or a humoral immune response. The maturation pathway chosen by the Th0 cell is often decisive for the outcome of disease and depends among others on the (co-)stimulatory attributes of the APC and the nature and abundance of cytokines provided by the APC and the microenvironment. In this study, we used macrophages, loaded ex vivo with antigen, for inciting Th0 activation and differentiation in vivo. The macrophages were derived from a clonal, immortalized population that both functionally and phenotypically expressed features characteristic of mature macrophages. Injection into syngeneic mice of IFN-gamma-treated, Ag-loaded macrophages induced a primary T cell response, indicated by the occurrence of a proliferative response in vitro after restimulation of splenocytes with Ag. Analysis of the accompanying cytokine secretion revealed high numbers of IFN-gamma-producing Th1 cells and only a few IL-4-secreting Th2 cells. This dominance of Th1 cells had functional implications, reflected in the high titer of Th1 cell-dependent IgG2 Abs and the absence of IgG1, characteristic of humoral immunity. Moreover, administration of Ag-loaded macrophages to mice with an ongoing Th1/Th2 response resulted in a complete suppression of IgG1 production, whereas IgG2 levels remained unaffected. These results demonstrate that macrophages exert APC activity in the organism, strongly skew primary responses to cellular immunity, and in addition suppress an already generated Th2-dependent humoral response, thus characterizing these cells as Th1-oriented APC.

Cytoglobin expression is upregulated in all tissues upon hypoxia: an in vitro and in vivo study by quantitative real-time PCR.

The vertebrate globin family has been extended with two members: neuroglobin and cytoglobin. We here investigate the changes of expression levels upon hypoxia of cytoglobin in parallel with neuroglobin, in vivo and in vitro, by using real-time quantitative PCR. Our data prove that cytoglobin is upregulated upon hypoxia in all tissues. The mechanism of induction of cytoglobin is regulated by the hypoxia-inducible factor 1, a posttranscriptionally regulated transcription factor controlling several hypoxia-inducible genes. The latter is argumented by: (1) cytoglobin is significantly upregulated upon hypoxia and this is dependent on the tissue and severity of hypoxia; (2) the regulation of cytoglobin expression in HIF-1 (+/-) knockout mice is affected; (3) the variations of the expression regulation are in the same manner as seen in the expression of our control gene VEGF, that is proven to be regulated by the HIF-1-pathway; and (4) cytoglobin promoter region contains HRE sites.

Quiescence-inducing and antiapoptotic activities of IL-15 enhance secondary CD4+ T cell responsiveness to antigen.

IL-15 shows functional redundancy with IL-2 due to its usage of the beta and gamma c subunit of the IL-2R. Yet, the requirement of IL-15 for an IL-15R alpha chain for high affinity interaction and the separate cellular sources of IL-2 and IL-15 suggest divergent activities for both cytokines. We compared the growth-inducing and proapoptotic or antiapoptotic activities of IL-15 and IL-2 on mature CD4+ T lymphocytes in the presence or absence of TCR occupancy. We found that the nature of IL-15 activity was critically dependent on the activation status of the T cells. In the absence of TCR triggering, IL-15 did not exert the growth factor activity of IL-2, but induced a quiescent phenotype, characterized by maintenance of the cells in the G0/G1 phase of the cell cycle and down-regulation of CD25, CD71, and CD95 expression. In the presence of appropriate TCR engagement, the IL-15-induced quiescent T cells were resistant against TCR-induced cell death and proliferated strongly. IL-2-treated cells, on the contrary, were sensitized to cell death, resulting in a negative feedback on cellular expansion and weak proliferative responsiveness. Consecutive action of IL-15 during the distinct phases of an in vitro immune response markedly increased the cell output of a second antigenic stimulation, as compared with IL-2. These results imply that during immune reactivity in vivo, IL-15 may take over from the transiently available IL-2 the role of survival factor but not of growth factor, hence promoting the long term maintenance of resting, Ag-experienced CD4+ T cells.

Interleukin-15 redirects the outcome of a tolerizing T-cell stimulus from apoptosis to anergy.

Clonal deletion and anergy are 2 mechanisms used by the immune system to establish peripheral tolerance. In vitro, these mechanisms are induced in T lymphocytes by triggering the T-cell receptor (signal 1) in the absence of costimulation (signal 2). T-cell clones have been shown either to become anergic or to die in response to signal 1 alone; yet the factors that govern this choice remain unknown. This study evaluated the influence of the cytokines interleukin (IL)-2 and IL-15 on the response of the Th1 clone hemagglutinin (T-HA) to signal 1, delivered by stimulation with immobilized anti-CD3 monoclonal antibody (mAb). The response induced by immobilized anti-CD3 mAb was dependent on the cytokine milieu; in the presence of IL-2, T-HA cells were subject to apoptosis, whereas in the presence of IL-15 the cells remained viable but showed proliferative unresponsiveness. After release from the anti-CD3 stimulus, the IL-15-rescued T-HA cells regained responsiveness to IL-2 and IL-15 growth factor activity. However, they were unable to proliferate when stimulated with their cognate antigen presented by professional antigen-presenting cells (signal 1 plus 2) and thus had acquired an anergic phenotype. These data assign a novel function to the previously reported antiapoptotic activity of IL-15, namely, the capacity to redirect the T-cell response to partial stimulation from clonal deletion to anergy. Furthermore, they emphasize that the cytokine environment can critically influence the outcome of a tolerizing stimulus.

Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo.

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.