Immunological biomarkers in patients with radiographic axial spondyloarthritis, an exploratory longitudinal Swedish study.

Objectives There is a need for more specific biomarkers to diagnose and predict disease course in patients with axial spondyloarthritis (axSpA). This study aimed to study immunological plasma biomarkers, at different time-points in radiographic (r)-axSpA patients overall and stratified by sex and compare these biomarker pattern in r-axSpA patients concerning disease phenotypes and disease activity. Methods Plasma samples were analysed from r-axSpA patients at and prior (Pre-Backbone) inclusion in the Backbone study. Interferon gamma, interleukin-10, -17A, -17F, -22, -23, -6, MCP-1, TNF-α, VEGF-A, MIF, IgA anti-CD74, zonulin, ESR, hsCRP, white blood cell count, and blood lipids were measured. Results Biomarker pattern discriminated significantly between r-axSpA patients in Backbone and Pre-Backbone compared with controls. When stratifying by sex, it was possible to discriminate between male and female r-axSpA patients in Backbone vs controls and between male r-axSpA patients in pre-Backbone and controls. In Backbone, markers with high discriminative capacity were MIF, IgA anti-CD74, and MCP-1. In Pre-Backbone, IL-6, TNF-α, MIF, triglycerides, cholesterol, IL-10, and zonulin displayed high discriminative capacity. Conclusion Based on their temporal pattern and mutual relationship, we suggest studying MIF, IgA anti-CD74, and MCP-1 in depth, at more time points, to further elucidate disease-driving mechanisms in this complex disease.

Increased carotid intima-media thickness in patients with radiographic axial spondyloarthritis compared to controls and associations with markers of inflammation.

OBJECTIVE: There is an increased risk for cardiovascular disease (CVD) in patients with radiographic axial spondyloarthritis (r-axSpA). In this cross-sectional study, we aimed to, overall and stratified by sex, (i) compare ultrasound derived carotid intima media thickness (cIMT), between patients and controls, and (ii) investigate associations between cIMT, clinical disease activity and inflammation-related laboratory markers in patients with r-axSpA. METHOD: In total, 155 patients diagnosed with r-axSpA using the modified New York criteria and 400 controls were included. Bilateral carotid ultrasound, laboratory testing, and questionaries were acquired. Disease-specific assessments were carried out for patients. Linear regression analysis was used to assess associations. RESULTS: Linear regression analyses showed that patients with r-axSpA had increased mean cIMT compared to controls (mean ± SD, 0.8 ± 0.1 mm vs 0.7± 0.1 mm, respectively, unstandardized ß (95% CI) -0.076 (-0.10, -0.052), P < 0.001) adjusted for smoking status and age. Linear regression analyses for patients with r-axSpA showed that only males presented significant associations between cIMT and inflammation-related laboratory markers, white blood cell (WBC) count (mean ± SD, 6.8 ± 1.6 109/L) and monocytes (0.6 ± 0.2 109/L); WBC count (unstandardized ß (95% CI) 0.019 (0.0065, 0.031), P = 0.003, R2 = 0.57) and monocytes (0.13 (0.0047, 0.26), P = 0.041, R2 = 0.55), adjusted for age, smoking status, body mass index, hypertension, dyslipidemia, diabetes mellitus, ASDAS-CRP, and treatment with DMARDs and glucocorticoids. No significant association was found between cIMT and clinical disease activity assessed by ASDAS-CRP. CONCLUSION: Patients with r-axSpA had significantly increased cIMT compared to controls. In male patients, higher WBC and monocyte count were associated with an increase in cIMT suggesting the role of inflammation in the development of atherosclerosis. Key Points •Carotid intima-media thickness was increased in patients with radiographic axial spondyloarthritis compared to controls. •White blood cell and monocyte counts were associated with carotid intima-media thickness in male patients with radiographic axial spondyloarthritis.

Protein Profiling in Presymptomatic Individuals Separates Myeloperoxidase-Antineutrophil Cytoplasmic Antibody and Proteinase 3-Antineutrophil Cytoplasmic Antibody Vasculitides.

OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a chronic relapsing condition with unknown etiology. To gain insight into the molecular processes underlying the disease, we examined biomarkers in blood samples collected prior to symptom onset. METHODS: The National Patient Register and Cause of Death register were searched for AAV-related International Classification of Diseases, Ninth Revision and Tenth Revision codes and linked to the registers from 5 biobanks. Eighty-five AAV patients with samples predating symptom onset of AAV were identified. For each case of AAV, 2 matched controls were included. Proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA expression levels were analyzed using enzyme-linked immunosorbent assays. Using an Olink Inflammation panel, 73 of 92 proteins were included after quality control. Data were replicated in a second cohort of 48 presymptomatic individuals and 96 controls. RESULTS: Of the 20 proteins with the lowest P values in the original cohort, 7 were replicated in the second cohort and 5 proteins were found to be significant between the groups in a meta-analysis. Eleven different pathways were identified in network enrichment analyses and were found to be significant in both cohorts. Stratification of samples obtained ≤5 years before symptom onset showed significant levels of CCL23, vascular endothelial growth factor A, and hepatocyte growth factor, which were also increased at borderline significant levels in the replication cohort (interleukin-6 was found to be significantly increased in the replication cohort). In presymptomatic AAV patients, 6 proteins were associated with MPO-ANCA positivity, and 7 proteins were associated with PR3-ANCA positivity. CONCLUSION: To our knowledge, this is the first study to identify protein markers preceding symptom onset in AAV patients. These findings set the stage for further research into the underlying cellular and molecular mechanisms in the pathogenesis of AAV and the diversification of patients into PR3-ANCA+ and MPO-ANCA+ subphenotypes.

HSC70 is a novel binding partner involved in the capture of immunoglobulins on B cells in the NOD mouse.

B cells have been shown to be essential for Type 1 diabetes development in the non-obese diabetic mouse, where their contribution as antigen presenting cells has been emphasised. Other important functions for B cells include surface capture of immunoglobulins and transportation of immune complexes, with subsequent endocytosis, antigen processing and antigen presentation. We have previously demonstrated that NOD B cells capture IgM and IgG immune complexes through an unknown surface molecule. In this study, we revealed the presumptive immunoglobulin-binding molecule to be HSC70. Moreover, we detected increased levels of HSC70 on NOD B cells. HSC70 has been shown to play a role in antigen processing and presentation as well as being important in several autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus. Due to its protein stabilising properties, increased HSC70 could contribute to enhanced self-antigen collection and presentation and thereby contribute to the development of Type 1 diabetes.

Increased proportions of inflammatory T cells and their correlations with cytokines and clinical parameters in patients with ankylosing spondylitis from northern Sweden.

Ankylosing spondylitis (AS) is an autoimmune disease affecting parts of the skeletal structure in particular. Previously increased levels of the inflammatory cell types Th17, Th22, Tc17 and Tc22 cells have been shown to be associated with AS. Here, we analysed the levels of inflammatory T cell subsets, related cytokines and clinical characteristics of AS patients vs controls from northern Sweden. Peripheral blood mononuclear cells (PBMCs) obtained from 50 AS patients and 50 matched controls were short term stimulated with PMA/Ionomycin, stained and analysed by flow cytometry. Plasma levels of Interleukin (IL)-17, IL-22, IL-10 as well as clinically relevant markers were determined. Compared to male controls, male AS patients showed 1.5- to 2-fold increases of Th17 (P = .013), Th22 (P = .003) and Tc22 (P = .024) among CD45+ CD3+ lymphocytes. Plasma IL-22 levels correlated with the Tc17 proportion in male patients (Rs  = 0.499, P = .003) and plasma IL-10 levels were inversely correlated with Tc17 among all patients (Rs  = -0.276, P = .05). Male patients with syndesmophytes showed significantly higher Th17 proportions (P = .038). In female AS patients, Tc22 was negatively correlated with C-reactive protein (high sensitivity) (hsCRP) (Rs  = -0.573, P = .016). We confirmed increased proportions of inflammatory T cells and correlations with relevant cytokines from male AS patients. The correlation between Th17 and syndesmophytes supports a role of Th17 in the pathogenic process.

Anti-CD74 IgA autoantibodies in radiographic axial spondyloarthritis: a longitudinal Swedish study.

OBJECTIVES: Antibodies against anti-CD74 are related to axial spondyloarthritis (axSpA). The objectives were (i) to study IgA anti-CD74 in radiographic (r)-axSpA patients in the Backbone cohort and to calculate the sensitivity and specificity of anti-CD74, (ii) to study the fluctuation of IgA anti-CD74 levels in prospectively collected samples, and (iii) to explore the relation between IgA anti-CD74 and radiographic spinal changes. METHODS: IgA anti-CD74 was analysed by ELISA in 155 patients with r-axSpA and age- and sex-matched controls. BASDAI, ASDAS, BASFI and BASMI were assessed and spinal radiographs were scored for r-axSpA-related changes with mSASSS. Previously donated samples, before inclusion in the Backbone study, were identified in the Medical Biobank of Northern Sweden. RESULTS: A total of 155 patients comprising 69% men and 31% women, age [mean (s.d.)] 55.5 (11.4) years and 152 (98.1%) HLA-B27 positive, were included. The plasma level of IgA anti-CD74 was significantly higher in the patients [median (interquartile range), 12.9 (7.9-17.9) U/ml] compared with controls [10.9 (7.2-14.6) U/ml, P = 0.003]. IgA anti-CD74 was above the cut-off level of 20 U/ml in 36/155 (23.2%) patients and in 15/151 (9.9%) controls (P = 0.002). Multivariable logistic regression analyses revealed ≥1 syndesmophyte associated with IgA anti-CD74 (odds ratio 5.64; 95% CI: 1.02, 35.58; P = 0.048) adjusted for hsCRP, smoking, BMI, sex and age. No distinct pattern of IgA anti-CD74 over time was revealed. CONCLUSION: Plasma levels of IgA anti-CD74 were increased in r-axSpA and independently associated with radiographic spinal changes, which suggests that IgA anti-CD74 could play a role in the pathogenies of r-axSpA.

Atherosclerosis in rheumatoid arthritis: associations between anti-cytomegalovirus IgG antibodies, CD4+CD28null T-cells, CD8+CD28null T-cells and intima-media thickness.

OBJECTIVES: Patients with rheumatoid arthritis (RA) have an accelerated progression of atherosclerosis. The aims of this study were to study the associations between subsets of T-cells, subclinical atherosclerosis assessed by intima-media thickness (IMT) and serological status for CMV in patients with RA. METHODS: Patients with new-onset RA (n=79), aged ≤60 years at diagnosis, were included in a prospective study of atherosclerosis. Controls matched for age and sex were also included (n=44). Ultrasound measurement of IMT in the common carotid artery was undertaken at inclusion (T0), after 1.5 years (T1.5) and after 11 years (T11). At T11, flow-cytometry analysis was undertaken to investigate subsets of T-cells. Serological analysis for CMV was undertaken from samples collected at T0. RESULTS: At T0, 66% of the patients and controls were CMV immunoglobulin G-positive. CMV-IgG positive patients had a significantly more rapid increase in IMT at T1.5, compared with controls and CMV-IgG negative patients. CMV-IgG positive patients had a significantly higher percentage of T-cells lacking CD28 (both CD4+CD28null and CD8+CD28null T-cells) than CMV-IgG negative patients. Increased levels of CD4+CD28null and CD8+CD28null T-cells were significantly associated with IMT at T11, adjusted for systolic blood pressure. CX3CR1 was expressed in CD4+ and CD8+ CD28null T-cells, but CX3CR1 per se was not associated with increased IMT. CONCLUSIONS: Presence of CMV IgG-antibodies in patients with RA is associated with altered T-cell-populations and an increased burden of atherosclerosis. A possible protective effect of antiviral treatment in CMV-positive patients with new-onset RA should be considered.

Dysregulated microRNA expression in rheumatoid arthritis families-a comparison between rheumatoid arthritis patients, their first-degree relatives, and healthy controls.

OBJECTIVE: Recent studies have demonstrated an altered expression of certain microRNAs in patients with rheumatoid arthritis (RA) as well as their first-degree relatives (FDRs) compared to healthy controls (HCs), suggesting a role of microRNA in the progression of the disease. To corroborate this, a set of well-characterized RA families originating from northern Sweden were analyzed for differential expression of a selected set of microRNAs. METHOD: MicroRNA was isolated from frozen peripheral blood cells obtained from 21 different families and included 26 RA patients, 22 FDRs, and 21 HCs. Expression of the selected microRNAs miR-22-3p, miR-26b-5p, miR-34a-3p, miR-103a-3p, miR-142-3p, miR-146a-5p, miR-155, miR-346, and miR-451a was determined by a two-step quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis including clinical variables was applied. RESULTS: Out of the nine selected microRNAs that previously have been linked to RA, we confirmed four after adjusting for age and gender, i.e., miR-22-3p (p = 0.020), miR-26b-5p (p = 0.018), miR-142-3p (p = 0.005), and miR-155 (p = 0.033). Moreover, a significant trend with an intermediate microRNA expression in FDR was observed for the same four microRNAs. In addition, analysis of the effect of corticosteroid use showed modulation of miR-103a-3p expression. CONCLUSIONS: We confirm that microRNAs seem to be involved in the development of RA, and that the expression pattern in FDR is partly overlapping with RA patients. The contribution of single microRNAs in relation to the complex network including all microRNAs and other molecules is still to be revealed. Key Points • Expression levels of miR-22-3p, miR-26b-5p, miR-142-3p, and miR-155 were significantly altered in RA patients compared to those in controls. • In first-degree relatives, a significant trend with an intermediate microRNA expression in FDR was observed for the same four microRNAs.

Protein profiling and network enrichment analysis in individuals before and after the onset of rheumatoid arthritis.

BACKGROUND: Antibodies and upregulated cytokines and chemokines predate the onset of rheumatoid arthritis (RA) symptoms. We aimed to identify the pathways related to the early processes leading to RA development, as well as potential novel biomarkers, using multiple protein analyses. METHODS: A case-control study was conducted within the Biobank of northern Sweden. The plasma samples from 118 pre-symptomatic individuals (207 samples; median predating time 4.1 years), 79 early RA patients, and 74 matched controls were analyzed. The levels of 122 unique proteins with an acknowledged relationship to autoimmunity were analyzed using 153 antibodies and a bead-based multiplex system (FlexMap3D; Luminex Corp.). The data were analyzed using multifactorial linear regression model, random forest, and network enrichment analysis (NEA) based on the 10 most significantly differentially expressed proteins for each two-by-two group comparison, using the MSigDB collection of hallmarks. RESULTS: There was a high agreement between the different statistical methods to identify the most significant proteins. The adipogenesis and interferon alpha response hallmarks differentiated pre-symptomatic individuals from controls. These two hallmarks included proteins involved in innate immunity. Between pre-symptomatic individuals and RA patients, three hallmarks were identified as follows: apical junction, epithelial mesenchymal transition, and TGF-ß signaling, including proteins suggestive of cell interaction, remodulation, and fibrosis. The adipogenesis and heme metabolism hallmarks differentiated RA patients from controls. CONCLUSIONS: We confirm the importance of interferon alpha signaling and lipids in the early phases of RA development. Network enrichment analysis provides a tool for a deeper understanding of molecules involved at different phases of the disease progression.

Innate lymphoid cells are present in gingivitis and periodontitis.

BACKGROUND: Innate lymphoid cells (ILCs) are the most recently identified leukocytes of the immune system and these cells are increasingly acknowledged to play important roles in host defence and tissue repair. ILCs are also contributors of inflammatory diseases such as asthma and colitis. We analyzed the presence and relative proportions of the different ILC subsets (ILC1, ILC2 and ILC3) in gingivitis and periodontitis. Further, we investigated if ILCs express receptor activator of nuclear factor kappa-B ligand (RANKL), a cytokine crucial for osteoclast differentiation and bone resorption. METHODS: We collected gingivitis and periodontitis soft tissue and characterized ILC subsets including RANKL expression in single-cell suspensions using flow cytometry. RESULTS: ILCs were detected both in gingivitis and periodontitis. The majority of ILCs, in both conditions, were ILC1s. Furthermore, RANKL expression was detected on a fraction of the ILC1s. CONCLUSIONS: Our discovery of the presence of ILCs both in gingivitis and periodontitis and concomitant expression of RANKL on a fraction of the ILC1 population suggest that these cells may be of importance in periodontal disease. In addition, our findings provide a new insight into the field of oral immunology.

Contribution of both B-cell intrinsic alterations as well as non-hematopoietic-derived factors in the enhanced immune response of the NOD mouse.

The underlying cellular and molecular mechanism for the development of Type 1 diabetes is still to be fully revealed. We have previously demonstrated that the NOD mouse, a model for Type 1 diabetes, display a prolonged and enhanced immune response to both self and non-self-antigens. The molecular explanation for this defect however, has not been determined. In this study we immunized NOD and C57BL/6 (B6) with the conventional antigen i.e. hen egg lysozyme (HEL) and analyzed B cell activation, germinal center reaction and antibody clearance. Corroborating our previous observations NOD mice responded robustly to a single immunization of HEL. Immunofluorescence analysis of the spleen revealed an increased number of germinal centers in unimmunized NOD compared to B6. However, post immunization germinal center numbers were similar in NOD and B6. NOD mice showed lower response to BCR stimulation with anti-IgM, in particular at lower concentrations of anti-IgM. Antibody clearance in vivo did not differ between the strains. To determine the cell type that is responsible for the prolonged and enhance immune response, we reconstituted NOD-RAGs with cells from primed donors in different combinations. NOD B cells were required to reproduce the phenotype; however the non-lymphoid compartment of NOD origin also played a role. Based on our results we propose that preexisting GCs in the NOD promote the robust response and alteration in the BCR signaling could promote survival of stimulated cells. Overall, this mechanism could in turn also contribute to the activation and maintenance of autoreactive B cells in the NOD mouse.

Elevated systemic glutamic acid level in the non-obese diabetic mouse is Idd linked and induces beta cell apoptosis.

Although type 1 diabetes (T1D) is a T-cell-mediated disease in the effector stage, the mechanism behind the initial beta cell assault is less understood. Metabolomic differences, including elevated levels of glutamic acid, have been observed in patients with T1D before disease onset, as well as in pre-diabetic non-obese diabetic (NOD) mice. Increased levels of glutamic acid damage both neurons and beta cells, implying that this could contribute to the initial events of T1D pathogenesis. We investigated the underlying genetic factors and consequences of the increased levels of glutamic acid in NOD mice. Serum glutamic acid levels from a (NOD×B6)F2 cohort (n = 182) were measured. By genome-wide and Idd region targeted microsatellite mapping, genetic association was detected for six regions including Idd2, Idd4 and Idd22. In silico analysis of potential enzymes and transporters located in and around the mapped regions that are involved in glutamic acid metabolism consisted of alanine aminotransferase, glutamic-oxaloacetic transaminase, aldehyde dehydrogenase 18 family, alutamyl-prolyl-tRNA synthetase, glutamic acid transporters GLAST and EAAC1. Increased EAAC1 protein expression was observed in lysates from livers of NOD mice compared with B6 mice. Functional consequence of the elevated glutamic acid level in NOD mice was tested by culturing NOD. Rag2-/- Langerhans' islets with glutamic acid. Induction of apoptosis of the islets was detected upon glutamic acid challenge using TUNEL assay. Our results support the notion that a dysregulated metabolome could contribute to the initiation of T1D. We suggest that targeting of the increased glutamic acid in pre-diabetic patients could be used as a potential therapy.

Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production.

B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACIhigh -expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse.

Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.

Lack of non-hematopoietic SIRPα signaling disturbs the splenic marginal zone architecture resulting in accumulation and displacement of marginal zone B cells.

Signal regulatory protein α (SIRPα) is an immunoglobulin super family protein predominantly expressed by myeloid but not lymphoid cells, and its role in lymphocyte homeostasis and function is still to be revealed. We demonstrate that mice bearing a mutant SIRPα lacking the cytoplasmic signaling domain (SIRPα MT) had an increased amount of splenic marginal zone (MZ) B cells compared to wild-type controls. Immunohistochemical analysis revealed an increased localization of MZB cells into B cell follicular areas of the white pulp in SIRPα MT spleens. However, we found no signs of an increased MZB cell activation level in MT mice. The immune response to T-independent antigens in vivo was slightly increased in SIRPα MT mice while sorted MZB from these mice responded normally to LPS in vitro. Bone marrow reconstitution experiments demonstrated that the MZB cell phenotype of SIRPα MT mice was due to lack of SIRPα signaling in non-hematopoietic cells. In contrast, MZ retention of MZ macrophages required hematopoietic SIRPα, while normal distribution of metallophilic macrophages required non-hematopoietic SIRPα signaling. In summary, these data identified SIRPα signaling in non-hematopoietic cells to play an important role in regulating the numbers and positioning MZB cell in the spleen.

Contribution of autoallergy to the pathogenesis in the NOD mice.

The immunoglobulin isotype IgE is commonly associated with allergy. However, its involvement in autoimmune disease in general, and Type 1 diabetes (T1D) in particular, is still not completely clarified, nonetheless IgE has been observed in patients with T1D. In this article, we aimed to elucidate the contribution of IgE in the pathogenesis of the disease in a spontaneous model for T1D, i.e. the NOD mouse. We observed increased levels of IgE in splenic, lymph node and peripheral blood B cells in the NOD mice compared to the control C57BL/6 (B6) mice. No correlation was found between the IgE levels on B cells and those in the sera of these mice, indicating a B cell intrinsic property mediating IgE capture in NOD. Functionally, the B cells from NOD were similar to B6 in rescuing the IgE-mediated immune response via the low affinity receptor CD23 in a transgenic adoptive transfer system. However, the involvement of IgE in diabetes development was clearly demonstrated, as treatment with anti-IgE antibodies delayed the incidence of the diabetes in the NOD mice compared to the PBS treated group. Pancreas sections from a 13-week-old NOD revealed the presence of tertiary lymphoid structures with T cells, B cells, germinal centers and IgE suggesting the presence of autoantigen specific IgE. Our study provides an insight to the commonly overlooked immunoglobulin IgE and its potential role in autoimmunity.

Altered metabolic signature in pre-diabetic NOD mice.

Altered metabolism proceeding seroconversion in children progressing to Type 1 diabetes has previously been demonstrated. We tested the hypothesis that non-obese diabetic (NOD) mice show a similarly altered metabolic profile compared to C57BL/6 mice. Blood samples from NOD and C57BL/6 female mice was collected at 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13 and 15 weeks and the metabolite content was analyzed using GC-MS. Based on the data of 89 identified metabolites OPLS-DA analysis was employed to determine the most discriminative metabolites. In silico analysis of potential involved metabolic enzymes was performed using the dbSNP data base. Already at 0 weeks NOD mice displayed a unique metabolic signature compared to C57BL/6. A shift in the metabolism was observed for both strains the first weeks of life, a pattern that stabilized after 5 weeks of age. Multivariate analysis revealed the most discriminative metabolites, which included inosine and glutamic acid. In silico analysis of the genes in the involved metabolic pathways revealed several SNPs in either regulatory or coding regions, some in previously defined insulin dependent diabetes (Idd) regions. Our result shows that NOD mice display an altered metabolic profile that is partly resembling the previously observation made in children progressing to Type 1 diabetes. The level of glutamic acid was one of the most discriminative metabolites in addition to several metabolites in the TCA cycle and nucleic acid components. The in silico analysis indicated that the genes responsible for this reside within previously defined Idd regions.

The prolonged and enhanced immune response in the non-obese diabetic mouse is dependent on genes in the Idd1/24, Idd12 and Idd18 regions.

In non-obese diabetic (NOD) mice B cells are an absolute requirement for T1D development. NOD mice display various B cell related immune deviations when compared to normal mice such as an enhanced and prolonged immune response towards several antigens, including non-self immunoglobulins. We hypothesized that this trait contributes to diabetes pathogenesis, and investigated the genetic factor(s) governing the altered immune response. A (NODxC57BL/6)F(2) cohort (n = 214) were analyzed for its primary immune response against a BALB/c derived monoclonal antibody, and a genome wide linkage analysis was performed. Significant linkage to the Idd1/Idd24, Idd12 and Idd18.1 regions as well as to a proximal region (marker D2Mit367, 33.5 Mb) on chromosome 2 was detected. We verified the observed linkage by analyzing a set of H2 congenic NOD and C57BL/6 mice and narrowed down the region to 8 Mb. Interaction between Idd1/24 and Idd12, as well as the novel locus on chromosome 2 was observed. However, the action by Idd18.1 was not influenced by any of the other loci. In addition to the known H2 I-Aß(g7) allelic variant of Idd1 in NOD, candidate gene analysis revealed a significant difference in the transcription of the H2-O/DO molecule. We hypothesize that multiple mechanisms contribute to the loss of immune response control, including that peptide loading on MHC class II in B cells of NOD is altered.

Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis.

OBJECTIVE: To identify whether cytokines, cytokine-related factors, and chemokines are up-regulated prior to the development of rheumatoid arthritis (RA). METHODS: A nested case-control study was performed in 86 individuals who had donated blood samples before experiencing any symptoms of disease (pre-patients) and 256 matched control subjects (1:3 ratio). In 69 of the pre-patients, blood samples were also obtained at the time of the diagnosis of RA. The plasma levels of 30 cytokines, related factors, and chemokines were measured using a multiplex system. RESULTS: The levels of several of the cytokines, cytokine receptors, and chemokines were significantly increased in individuals before disease onset compared with the levels in control subjects; i.e., those representing signs of general immune activation (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-1 receptor antagonist, and tumor necrosis factor), activation of Th1 cells (interferon-gamma, IL-12), Th2 cells (IL-4, eotaxin), Treg cells (IL-10), bone marrow-derived factors (IL-7, granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor), as well as chemokines (monocyte chemotactic protein 1 and macrophage inflammatory protein 1alpha). The levels were particularly increased in anti-cyclic citrullinated peptide antibody- and rheumatoid factor-positive individuals, and the concentration of most of these increased further after disease onset. The concentration of IL-17 in individuals before disease onset was significantly higher than that in patients after disease onset. Individuals in whom RA subsequently developed were discriminated from control subjects mainly by the presence of Th1 cells, Th2 cells, and Treg cell-related cytokines, while chemokines, stromal cell-derived cytokines, and angiogenic-related markers separated patients after the development of RA from individuals before the onset of RA. CONCLUSION: Individuals in whom RA later developed had significantly increased levels of several cytokines, cytokine-related factors, and chemokines representing the adaptive immune system (Th1, Th2, and Treg cell-related factors); after disease onset, the involvement and activation of the immune system was more general and widespread.

Enhanced capture of extramembranous IgM and IgG on B cells in the NOD mouse--implications for immune complex trapping.

Binding of various antibody isotypes to B cells through either FcgammaRs or complement receptors has been attributed to play several roles, e.g. in immune complex (IC) transportation and regulation of B cell receptor signaling. We have revealed a novel B cell intrinsic receptor for IgM and IgG which is present in C57BL/6 (B6) mice and is more abundant in non-obese diabetic (NOD) mice. As a consequence, the level of extramembranous IgG monomers and IgM pentamers on peripheral blood B cells from NOD mice was significantly higher compared with B6 mice. The effect of this aberration was that all B cells in peripheral blood of (NOD.IgH(a) x B6(IgH(b)))F(1) mice carried both IgM allotypes on their surface. In addition, analysis of IC binding using IgG- or IgM-opsonized bacterial particles revealed a higher degree of binding in NOD mice compared with B6. We hypothesize that this novel Ig-binding receptor is part of the normal immune system function. The aberrant function in the NOD mouse could contribute to the development of Type 1 diabetes by altering normal B cell functions such as activation, IC transportation and B cell homeostasis.