Innate stimulation of B1a cells enhances the autoreactive IgM repertoire in the NOD mouse: implications for type 1 diabetes
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We sought to determine whether the presence of natural autoreactive antibodies of B1a cell origin would play a role in the initiation of type 1 diabetes.
We compared IgM repertoires and B1a cell compartments in NOD and C57BL/6 mice. Serum IgM autoreactivity profiles were determined by ELISA and the secretory properties and activation status of B1a cells were characterised by enzyme-linked immunosorbent spot (ELISPOT) assay and flow cytometry. B1a cell response to innate activation was analysed by gene expression assays, ELISA and [3H]thymidine incorporation. The effect of NOD IgM produced by B1a cells on NOD.severe combined immunodeficient (SCID) beta cells was examined in co-cultures: IgM binding was measured by flow cytometry and real-time PCR was used to study oxidative stress responses.
NOD mice displayed increased levels of serum anti-insulin IgM that were independent of the H2 locus, that were maintained up to prediabetic stages and that correlated with the NOD B1a cell secretion profile. NOD B1a cells had a naturally increased pattern of activation, expressed higher levels of toll-like-receptors (Tlrs) and responded to TLR stimulation in vitro with higher proliferation and increased capacity to secrete anti-type-1-diabetes-related IgM, but produced lower amounts of IL10. IgM of NOD B1a cell origin was able to bind to pancreatic beta cells in vitro and induce expression of inducible nitric oxide synthase (Nos2).
NOD B1a cells had a lower innate activation threshold for secretion of autoreactive IgM capable of triggering oxidative stress responses on binding to pancreatic beta cells; this provides an early mechanism that contributes to diabetes in a mouse model of type 1 diabetes.
KeywordsAutoreactive IgM B1a cells NOD Oxidative stress Pancreatic beta cell Toll-like receptor Type 1 diabetes
Islet cell antigen 512
Severe combined immunodeficient
Systemic lupus erythematosus
The urgency to identify the subtle factors contributing to type 1 diabetes onset and progression is compelled by recent European estimates indicating that the number of new cases of type 1 diabetes in children under the age of 5 years is increasing by more than 5% each year and predicting the doubling of its incidence in this age group by 2020 . Patients with type 1 diabetes and NOD mice, which are a model of type 1 diabetes, share the spontaneous pathogenic events of type 1 diabetes development  in which autoreactive cytotoxic T cells selectively destroy insulin-secreting beta cells within the pancreatic islets of Langerhans . Nevertheless, studies on NOD mice devoid of B cells have shown that this lymphocyte population is necessary in the diabetic autoimmune process  and it is widely accepted that islet-related autoantibodies (AAbs) represent the earliest manifestations of diabetogenesis in both prediabetic patients and in NOD mice . These autoreactive immunoglobulins, of the IgG isotype, recognise beta cell antigens and are believed to be bystander products of the disease resulting from an ongoing autoimmune process . Thus, at the stage of cytotoxic beta cell destruction, B cells would be highly exposed to beta cell-related autoantigens (AAgs), leading to preferential activation of B cells with autoreactive potential. In fact, NOD B cell receptor specificity and the capacity of B cells to present antigens to T cells are determinant factors in type 1 diabetes progression [6, 7]. It is believed that long-sustained autoreactive T–B cell interactions condition the repertoire of AAbs typical of prediabetic patients, in whom the beta cell destruction is already in progress, though clinical signs are not yet present. Importantly, B cell-null NOD mice that express a transgene encoding a mutant heavy chain immunoglobulin on the cell surface but that cannot secrete antibodies only partially restored type 1 diabetes, suggesting that circulating antibodies could play a role in the disease .
Here, we revisited the role of AAbs in type 1 diabetes pathogenesis with the contention that the early presence of AAbs against beta cell antigens can have implications for type 1 diabetes pathogenesis. Insulitis consists of the infiltration of pancreatic islets of Langerhans by mononuclear cells and is the hallmark of type 1 diabetes initiation. In NOD mice the first insulitic events start at about 3–5 weeks of age but clinical signs of diabetes, due to massive beta cell loss, arise only from 12 weeks onwards . It has previously been shown that NOD mice, but not C57BL/6 non-diabetic control mice, have antibodies bound to beta cells before the development of insulitis. Interestingly, these antibodies are mainly of the IgM isotype  and thus are unlikely to originate in germinal centre reactions. Consistently, young NOD mice have a pronounced repertoire of IgM AAbs . Most of the antibodies of the IgM isotype are spontaneously secreted by B1a cells in the absence of exogenous stimuli or T cell help . The B1a cells are part of the B1 cell subset of lymphocytes that are mainly found in the peritoneal and pleural cavities and at lower levels in the spleen. These B1 cells express low levels of B220 and are subdivided into B1a and B1b subsets based on the presence or absence of the CD5 molecule on their surface, respectively. B1a cells are mostly of fetal origin, do not undergo class switching and affinity maturation and spontaneously secrete natural AAbs . On the other hand, B1b cells are generated mainly during neonatal life in the bone marrow  and are involved in the earliest specific reactions against pathogens [15, 16]. CD5 expression on B1a cells is known to downmodulate responses to B cell-receptor-mediated signalling while favouring IL10 secretion [17, 18]. Interestingly, the B1 cell activation status is influenced by gut microflora modifications [19, 20] and innate stimulation through toll-like receptors (TLRs) can induce their proliferative and antibody-secretion capacities . Thus, B1 can be considered as bridging cells between innate and adaptive immunity.
Importantly, B1a cells have recently been proven necessary for insulitis onset in a transgenic model in which disease is mediated by T cells recognising pancreatic beta cell antigens . Also, in the 125Tg mouse, B1a cells prevailed as anti-insulin IgM secretors while B2 cells with the same specificity were anergised . Further, B1a cells are increased in the peripheral blood of type 1 diabetes patients  and have been suggested to play a role in the NOD mouse autoimmune process .
We have previously hypothesised that natural antibodies (NAbs) of NOD B1a cell origin could promote the initiation of insulitis . However, the link between B1a cell properties, autoreactive IgM profile and autoimmune process triggering has not been established for type 1 diabetes. Here, we dissect this intricate chain of events by analysing the autoreactive repertoire of NOD IgM, by uncovering the B cell subpopulations contributing to its secretion and by examining the impact of these immunoglobulins on NOD beta cell physiology.
C57BL/6, NOD, NOD.severe combined immunodeficient (SCID), C57BL/6.H2g7 and NOD.H2b mice, 1–12 weeks of age, were bred and maintained in specific-pathogen-free facilities at the Instituto Gulbenkian de Ciência. Insulitis was rare at 3 weeks in NOD female mice (electronic supplementary material [ESM] Fig. 1). Protocols were approved by the competent Portuguese authority, in accordance with international regulations .
ELISA and ELISPOT assays
ELISAs were used to quantify total IgM and anti-insulin IgM concentrations in mouse sera. Total IgM, IgM with reactivity to a pool of type-1-diabetes-related AAgs, namely insulin, islet cell antigen 512 (IA-2), glutamate decarboxylase (GAD)65 and GAD67 and IL10 levels were measured in cell culture supernatant fractions after TLR stimulation (see ESM Methods).
The number of B cells secreting IgM with reactivity to type 1 diabetes AAgs, thyroiditis-associated AAgs (thyroglobulin and thyroperoxidase), muscarinic-3-receptor (sialitis-related AAg), systemic lupus erythematosus (SLE)-associated AAg (double-stranded [ds]DNA, single-stranded [ss]DNA and histone) or AAg recognised by B1a cells (phosphatidylcholine, phosphorylcholine and dextran)  were determined as described elsewhere  (see ESM Methods).
Cell analysis and purification
Cell suspensions prepared from the spleen and peritoneal cavity were incubated with anti-mouse-Fc-block/CD16/32 (clone 2.4 G2, Becton Dickinson, Franklin Lakes, NJ, USA). Peritoneal B1a cells were identified as B220+CD5+ and B1b + B2 cells as B220+CD5−. Splenic B1a cells were identified as CD23−IgMhighB220lowCD5+, follicular B lymphocytes as CD23+IgMlow and marginal zone B cells as CD23−IgMhighCD21+CD5−. Fluorescein isothiocyanate (FITC)-anti-CD19 (clone 1D3), FITC-anti-IgM (clone R33.24.12), phycoerythrin (PE)-anti-CD23 (clone B3B4), peridinin-chlorophyll protein (PerCP) or PE-anti-CD5 (clone 53-7.3), allophycocyanin (APC)-anti-CD45R/B220 (clone RA3-6B2), biotinylated or FITC-anti-CD11b/macrophage-1 antigen (MAC-1) (clone M1/70), FITC-anti-CD21 (clone 7 G6), PE-anti-CD43 (clone S7), biotinylated-anti-CD86 (clone GL1), FITC-anti-CD62L (clone Mel14), biotinylated or PE-anti-syndecan-1 (clone 281-2) and FITC or PerCP-streptavidin antibodies were used for staining. Traceable beads (Beckman Coulter, Brea, CA, USA) were added for counting cells. Flow cytometry (FACSCalibur) was used and analysis performed with FlowJo software (TreeStar, Ashland, OR, USA). Peritoneal and splenic B cells were purified by cell sorting using MoFlo (Dako-Cytomation, Berkeley, CA, USA) or BD FACSAria III cell sorter (Becton Dickinson) with purity above 90%.
RNA isolation and real-time PCR
Total RNA from sorted peritoneal B1a cells or from cultured beta cells was obtained using a High Pure RNA Isolation Kit (Roche, Basel, Switzerland) following the manufacturer’s protocol. RNA was converted to cDNA with Transcriptor High Fidelity cDNA Synthesis Kit (Roche). The following TaqMan gene expression assays with FAM reporter (Applied Biosystems, Foster City, CA, USA) were used: Tlr2 (Mm00442346_m1), Tlr4 (Mm00445274_m1), Tlr6 (Mm02529782_s1), Tlr7 (Mm00446590_m1), Tlr9 (Mm00446193_m1), Fas (Mm00433237_m1), Nos2 (Mm01309901_m1), Casp3 (Mm01195084_m1) and Ccl2 (Mm00441242_m1). Gene expression was quantified in ABI Prism 7900HT (Applied Biosystems). Relative quantification was obtained after normalisation for mouse GAPDH (VIC/MGB probe) expression using the 2ΔΔC t analysis method .
Purified peritoneal B1a cells were cultured in RPMI 1640 complete medium (Gibco, Grand Island, NY, USA) with or without 1–5 μg/ml purified lipolysaccharide (LPS) (Invivogen, San Diego, CA, USA) and supernatant fractions taken for antibody and IL10 quantification by ELISA at day 3 of culture. For proliferation analysis, cells were pulsed in the last 6 h of culture, harvested and [3H]thymidine (Perkin Elmer, Waltham, MA, USA) incorporation measured. Expression of activation markers after 1 day of stimulation was assessed on pooled duplicates using biotinylated anti-CD69 (clone H1.2 F3), biotinylated anti-CD86 (clone GL1), Alexa488-anti-CD25 (clone PC 61) and PerCP-streptavidin.
In vitro transwell cultures
Islets of Langerhans were isolated from the pancreas of 9-week-old NOD.SCID female mice by collagenase type V digestion (1.4 mg/ml; Sigma-Aldrich, St Louis, MO, USA) followed by hand picking under a stereomicroscope. Islets were dissociated into single cell suspensions by mechanical and dispase enzymatic treatment (5 mg/ml; Roche). Freshly dissociated cells were cultured at 1 × 105 cells per well in Ham’s F-10-glutamax medium (Gibco) with 10 mmol/l glucose (Sigma-Aldrich), 0.5% BSA, 50 μmol/l isobutylmethylxanthine (Sigma-Aldrich), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco). At 24 h after this treatment, sorted peritoneal B1a cells (5 × 105) or follicular splenic B cells (5 × 105) were added in transwells (0.4 μm pore size; Millipore, Billerica, MA, USA). After 24 h of co-culture beta cells were collected for flow cytometry or RNA isolation. Alexa647-anti-IgM (clone R33.24.12) was used for staining of bound IgM on beta cells. Negative controls consisted of beta cells with lymphocyte medium only in the transwells.
Statistics were estimated by either Kruskal–Wallis or unpaired Student’s t test as specified in figure legends. Two-tailed tests with 95% confidence intervals were used and differences with p < 0.05 were considered significant.
High anti-insulin IgM level in the NOD mouse is independent of T cell autoreactivity
Additionally, we have analysed the global pattern of natural IgM reactivity by comparing the capacity of NOD and C57BL/6 serum IgM to recognise AAg present in NOD protein extracts and inferred that not only were there different patterns of IgM autoreactivity but also that global IgM reactivity was higher in NOD compared with C57BL/6 mice (ESM Fig. 2). We have also measured the serum levels of anti-insulin IgG in NOD and C57BL/6 mice. As expected, the reactivity profile was quite different from that observed for anti-insulin IgM. Although very low to undetectable levels were present in the serum of both strains up to 6 weeks, NOD mice had a progressive increase in antibody concentration that was most relevant at 12 weeks (Fig. 1d).
High proportion of NOD peritoneal cavity B1a cells secretes anti-type 1 diabetes IgM in absence of patent insulitis
We next wanted to ascertain whether the observed difference in autoreactivity was restricted to type-1-diabetes-associated AAg or whether it could represent a general feature of NOD B1a cells. Thus, we have quantified the proportion of B1a cells secreting IgM recognising either a pool of AAg related to autoimmune thyroiditis (Fig. 2b), the main self-antigen of immune-mediated sialitis (Fig. 2c), a pool of antigens that are targets of SLE (Fig. 2d) or a pool of antigens that B1a cells generally recognise (Fig. 2e). Consistent with the NOD polyendocrine phenotype we detected more peritoneal B1a cells secreting IgM that recognised thyroiditis and sialitis AAgs (Fig. 2b,c), although no differences were found for the other AAgs tested (Fig. 2d,e).
NOD B1a cells are B220bright at 1 week of age
Peritoneal cavity NOD B1a cells display increased expression of surface activation molecules
NOD B1a cells secrete increased levels of autoantibodies on TLR stimulation
IgM secreted by NOD B1a cells can bind to pancreatic beta cells in vitro and trigger Nos2 expression
This study demonstrates that NOD B1a cells have an increased responsiveness to innate activation and secrete NAbs with higher reactivity to type-1-diabetes-associated AAg. Importantly, NOD-B1a-cell-derived IgM is able to bind pancreatic beta cells and trigger Nos2 expression, a starting point in the beta cell oxidative stress response. Together, these results provide evidence to support the proposal that the NOD natural AAb repertoire is biased from an early age towards endocrine autoreactivity, thus having the potential to fuel the autoimmune beta cell attack prior to the T-cell-mediated destruction.
In our time-course analysis NOD mice presented increased but stable serum levels of IgM with insulin reactivity when compared with C57BL/6 mice. This pattern of natural antibody autoreactivity did not correlate with the IgG serum levels that clearly followed the progression of the disease in the NOD mouse (Fig. 1d and Koczwara et al ). The remarkably different trajectories of serum anti-insulin IgG or IgM isotypes highlight their different roles in the NOD immune response. While the detection of anti-type-1-diabetes-AAg IgG is widely accepted as an early disease marker in clinical practice and is generally considered to be a by-product of the T-cell-mediated destruction of beta cells [32, 33], autoreactive IgM comprises the pool of NAbs, the first antibodies to arise during ontogeny, produced in the absence of exogenous stimuli . Analysis of MHC congenic strains  showed that, unlike IgG, IgM reactivity observed in the NOD cells is independent of T cell help and disease progression, and is consistent with the hypothesis that an increased autoreactivity in the NOD NAb compartment would contribute to the initiation of autoimmunity . This suggested the possibility that IgM reactivities would also be altered in human type 1 diabetes and we have preliminary evidence that common genetic variants (single-nucleotide polymorphisms) in the IgM locus control the levels of anti-GAD serum IgM and are associated with disease in a collection of type 1 diabetes patients (I. Rolim, G. Barata, J. Raposo, M. Catarino, C. Penha-Gonçalves, unpublished results). These findings strengthen the link between NAbs and type 1 diabetes pathogenesis and give importance to further investigations into the origin of autoreactive IgM.
We found that B220 expression strikingly distinguished NOD B1a cells in the peritoneal cavity from as early as 1 week of age, as compared with C57BL/6 and BALB/c cells (not shown). Interestingly, peritoneal B1a cells that develop from adult bone marrow progenitors showed increased levels of B220 expression [36, 37]. Thus, it is possible that bone marrow progenitors play a larger role in NOD B1a cell ontogeny, conditioning a higher B220 expression. Additionally, increased B220 expression is associated with a lower threshold of activation [38, 39] and possibly underlies the surface immunophenotypes observed in NOD peritoneal B1a cells. This B1a B cell surface profile was also observed in male NOD mice, strengthening the suggestion that such NOD phenotypes are genetically determined and not affected by the degree of beta cell destruction (ESM Fig. 4) . Some of these phenotypes have previously been described for NOD B1a cells and associated with increased activation, migration and capacity for T cell co-stimulation . Interestingly, alterations in the NOD microflora have been shown to condition some of the surface B1 cell traits [19, 20]. Thus, peritoneal cavity B1a cells are able to respond rapidly to external stimuli, namely microbial alterations in the intestine, leaving open the possibility that IgA-secreting B1a cells residing in the gut could influence the effect of microflora components on the development of diabetes in the NOD mouse . Our observations of higher B220 and Tlr expression at an early age and increased proliferation upon TLR4 stimulation concur to suggest strongly that B1a B220high cells may represent a B1 cell population in the NOD mouse that colonises the peritoneal cavity in early ontogeny and is exquisitely sensitive to innate stimuli.
In accordance, NOD B1a TLR stimulation resulted in increased levels of IgM secretion with reactivity against type 1 diabetes AAg while the secretion of IL10 remained lower when compared with C57BL/6 B1a cells. Stimulation through TLRs has been shown to strongly promote plasma cell differentiation in the B1 cell compartment . Interestingly, NOD B1a cells produced amounts of total IgM similar to those produced by C57BL/6 cells in the presence of TLR ligands. This indicates that the NOD B1a cell population harbours an increased population of autoreactive cells with increased responsiveness and propensity for plasma cell differentiation on innate stimulation. This is in agreement with increased numbers of NOD B1a cells secreting IgM with reactivity towards endocrine AAgs and with the pattern of serum anti-insulin IgM.
Importantly, we did not detect strain differences in the numbers of B1a cells in the pancreatic lymph nodes, nor their presence in the pancreas of prediabetic NOD mice (data not shown), strengthening the hypothesis that the role of NOD B1a cells on type 1 diabetes onset may be mediated by autoreactive IgM . Accordingly, binding of IgM to beta cells has been described in vivo in very young NOD mice . We showed here that NOD B1a-derived IgM binds to beta cells in vitro and that the expression of Nos2 is upregulated in islet cells in contact with NOD B1a-derived supernatant fraction, suggesting that IgM binding is involved in triggering the beta cell oxidative stress response. However, Fas, Casp3 and Ccl2 gene expression was not altered, indicating that the pattern of gene induction differed from that described for cytokine-induced beta cell damage . This reinforces the notion that the effects observed in Nos2 expression are due to the binding of autoreactive IgM, a hypothesis that requires further confirmation. In addition, the binding of IgM did not induce the expression of apoptosis-related genes, nor a decrease in beta cell viability as determined by vital staining at 24 h of culture (data not shown). Nevertheless, specific assays for apoptosis or longer co-culture periods would be necessary to demonstrate if IgM binding impacts on beta cell viability. Whether other immunological components, such as complement in the serum, could potentiate the IgM effect and determine the fate of beta cells in type 1 diabetes remains to be addressed.
In conclusion, in this study we have linked alterations in the B1a cell population to serum IgM autoreactivities and beta cell oxidative stress, strengthening the hypothesis that NAbs are an early factor in the evolution of diabetes pathogenesis in the NOD mouse model of type 1 diabetes.
We wish to thank D. Eizirik (Laboratoire de Médecine Expérimentale, Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium) for providing training on beta cell isolation and J. W. Thomas (Vanderbilt University Medical Center, Nashville, TN, USA) for kindly providing the anti-insulin IgM antibody. We are grateful to C. Fesel (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for help with the ‘Panama’ blot technique and R. M. Parkhouse (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for useful suggestions and discussions and to both of them for critically reading the manuscript.
We acknowledge Fundacão para a Ciência e a Tecnologia for financial support of J. Côrte-Real with grant SFRH/BD/29212/2006, and N. Duarte with grant SFRH/BPD/43631/2008.
CP-G and LT contributed to the conception and design of this study and critical reviewed the intellectual content of this manuscript. ND and JC-R contributed equally to the collection, analysis and interpretation of data as well as to the conception, design and drafting of this article. All authors approved the final version of the article to be published.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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