Toll-like receptor 9 negatively regulates pancreatic islet beta cell growth and function in a mouse model of type 1 diabetes
Innate immune effectors interact with the environment to contribute to the pathogenesis of the autoimmune disease, type 1 diabetes. Although recent studies have suggested that innate immune Toll-like receptors (TLRs) are involved in tissue development, little is known about the role of TLRs in tissue development, compared with autoimmunity. We aimed to fill the knowledge gap by investigating the role of TLR9 in the development and function of islet beta cells in type 1 diabetes, using NOD mice.
We generated Tlr9−/− NOD mice and examined them for type 1 diabetes development and beta cell function, including insulin secretion and glucose tolerance. We assessed islet and beta cell number and characterised CD140a expression on beta cells by flow cytometry. We also tested beta cell function in Tlr9−/− C57BL/6 mice. Finally, we used TLR9 antagonists to block TLR9 signalling in wild-type NOD mice to verify the role of TLR9 in beta cell development and function.
TLR9 deficiency promoted pancreatic islet development and beta cell differentiation, leading to enhanced glucose tolerance, improved insulin sensitivity and enhanced first-phase insulin secretory response. This was, in part, mediated by upregulation of CD140a (also known as platelet-derived growth factor receptor-α [PDGFRα]). In the absence of TLR9, induced by either genetic targeting or treatment with TLR9 antagonists, which had similar effects on ontogenesis and function of beta cells, NOD mice were protected from diabetes.
Our study links TLR9 and the CD140a pathway in regulating islet beta cell development and function and indicates a potential therapeutic target for diabetes prevention and/or treatment.
KeywordsCD140a Diabetes Islet beta cell PDGFRα TLR9
Intra-peritoneal glucose tolerance test
Insulin tolerance test
Platelet-derived growth factor
Platelet-derived growth factor receptor-α
Pancreatic and duodenal homeobox-1
The innate immune system generates early inflammatory responses to a variety of environmental insults. A large number of innate immune receptors, including the Toll-like receptors (TLRs), are important for immediate immune responses to infection, leading to later, more specific, adaptive immunity. On binding the appropriate ligand, the TLRs activate signalling pathways that lead to production of proinflammatory cytokines and upregulation of costimulatory molecules. TLRs were initially thought to be expressed mainly on immune cells, in particular antigen-presenting cells, but it is increasingly recognised that they are also expressed on many other cell types and have functions that range beyond activation of the immune system. We, and others, have shown that pancreatic beta cells express many TLRs in both mice and humans [1, 2]. Activation of TLR3, a receptor for double-stranded RNA, has been shown to induce beta cell apoptosis [1, 3, 4]. TLR4, the receptor for endotoxin, is involved in regulation of metabolism in a variety of tissues including beta cells [5, 6, 7]. TLR9 can also be detected easily in both mouse and human islets [1, 2].
Type 1 diabetes is a slowly progressing autoimmune disease. We, and others, have independently shown that TLR9-deficient (Tlr9−/−) NOD mice are protected from type 1 diabetes development [8, 9, 10]. This protection is mediated partly by impaired IFNα production from Tlr9−/− mouse dendritic cells  and by enhanced expression and regulatory function of CD73+ T cells . However, increasing evidence suggests that TLRs recognise not only exogenous ligands from microbes but also endogenous ligands from both normal and damaged cells. Recent studies suggest that DNA released from both physiological and pathological dying cells can be a key stimulus to innate immune activation of TLR9 [11, 12, 13, 14]. There is also evidence that TLRs regulate neurogenesis during development . Considering that islet beta cells undergo significant growth and remodelling, early in life [16, 17, 18, 19], it is likely that TLR9 plays an important role in the development of type 1 diabetes, beyond any direct immune function. However, to date, there have been no reports about the role of TLR9 in islet beta cell development. Therefore, we aimed to assess the role of TLR9 in the development and function of islet beta cells in both NOD and C57BL/6 mice.
All the mice used in the study were housed in specific pathogen-free conditions with a 12 h dark–light cycle and were housed in individually ventilated filter cages with autoclaved food and bedding at the Yale University animal facility. The Tlr9−/− NOD mice were generated by backcrossing Tlr9−/− C57BL/6 mice  with our NOD mice, for over 11 generations. The purity of the NOD genetic background was confirmed by mouse genome SNP scan with Illumina Infinium panel (DartMouse, Lebanon, NH, USA). Tlr9−/− NOD.Scid mice were generated by breeding Tlr9−/− NOD mice with NOD.Scid mice, which were originally purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained at Yale University for ~25 years. Wild-type (WT) C57BL/6 (Tlr9+/+ C57BL/6) mice were also purchased from the Jackson Laboratory and maintained at Yale University for ~10 years. The use of the animals in this study was approved by the IACUC of Yale University. All mice used in different experiments were randomly selected from different breeding cages and different litters. Experimenters were not blinded in this study.
Natural history of diabetes development
Tlr9−/− NOD mice and Tlr9+/+ NOD littermates were screened for glycosuria weekly for spontaneous diabetes development, up to 32 weeks of age. Diabetes was confirmed by blood glucose of ≥13.9 mmol/l with a FreeStyle glucose meter (Abbott, Chicago, IL, USA).
Streptozotocin-induced diabetes development
Female Tlr9−/− NOD mice and Tlr9+/+ NOD littermates (5–6 weeks old) were treated with either high-dose streptozotocin (STZ) (100 mg/kg, administered by two consecutive i.p. injections, 24 h apart) or low-dose STZ (40 mg/kg, administered by i.p. injection, once daily, for 5 days). Mice were screened for glycosuria daily for diabetes development and confirmed as above.
Intra-peritoneal glucose tolerance test
Intra-peritoneal glucose tolerance tests (IPGTTs) were performed in 5–6-week-old Tlr9−/− NOD, Tlr9+/+ NOD, Tlr9−/− C57BL/6, Tlr9+/+ C57BL/6, Tlr9−/− NOD.Scid and Tlr9+/+ NOD.Scid mice. The mice were fasted overnight with free access to water and the blood glucose was measured before (time zero) and after i.p. injection of glucose (1 g/kg) at different time points from blood samples. Blood glucose was measured by a FreeStyle glucose meter (Abbott). Data are shown from one out of three experiments, each confirming the significant difference.
Insulin tolerance test
Insulin tolerance tests (ITTs) were performed in 5–6-week-old male Tlr9−/− C57BL/6 mice and Tlr9+/+ C57BL/6 mice. The mice were fasted for 6 h with free access to water and the blood glucose was measured before and after i.p. injection of insulin (Humulin-R, 0.75 U/kg; Eli Lilly, Indianapolis, IN, USA) at different time points, as described for IPGTT.
Islet and beta cell isolation
Pancreatic islets were isolated as previously described . Mice were euthanised by cervical dislocation. The pancreas was inflated with 3 ml cold collagenase (Sigma; St Louis, MO, USA) solution (0.3 mg/ml) through the bile duct with a 20G needle starting at the gall bladder. The pancreas was then removed into a siliconised glass tube containing 2 ml of 1 mg/ml collagenase solution and digested at 37°C in a water bath for 12–15 min. After three washes of the digested pancreas, islets were hand-picked and counted under a dissecting microscope for further experiments. For single-cell isolation, the islets were treated with Cell Dissociation Solution (Sigma) and the single-cell suspension was harvested. Beta cells from the dissociated islets were stained with fluorochrome-conjugated monoclonal antibodies to CD45 (BioLegend; San Diego, CA, USA), CD140a (BioLegend) and FluoZin-3-acetoxymethyl (AM) (CD45−FluoZin-3-AM+; ThermoFisher, Waltham, ME, USA)  before being analysed by flow cytometry (LSRII; BD Bioscience, San Diego, CA, USA).
Pancreatic islets were isolated as described above. RNA from islets of 3–4-week-old female Tlr9+/+ NOD mice and Tlr9−/− NOD mice was extracted with an RNAeasy kit (Qiagen, Hilden, Germany) and quantified by NanoDrop (ThermoFisher). Equal amounts of RNA were reverse transcribed using SuperScript III First-strand synthesis kit with random hexamers (Invitrogen, Carlsbad, CA, USA). Quantitative PCR (qPCR) was performed using the Bio-Rad iQ5 qPCR detection system (Hercules, CA, USA) with the specific primers for Pdx-1 (also known as Pdx1) (5′-CAGCAGAACCGGAGGAGAAT-3′ and 5′-CGACGGTTTTGGAACCAGAT-3′) and Ngn3 (also known as Neurog3) (5′-CCCGCAGCTCTCTGTTCTTT-3′ and 5′-GGGTCTCTTGGGACACTTGG-3′) (Sigma). The relative expression of mRNA levels was determined with the 2−∆∆Ct method by normalisation with the housekeeping gene Gapdh (5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′).
Cell staining for flow cytometry
For direct staining, single-cell suspensions (~5 × 104 to 2 × 105 cells) of immune cells or islet cells were incubated with a 2.4G2 Fc-blocking antibody (10 mins, room temperature) prior to staining with pre-titrated amounts of monoclonal antibodies conjugated with different fluorochromes to combinations of CD3 (17A2), CD4 (GK1.5), CD44 (IM7), CD45 (30-F11) CD62L (MEL-14), CD140a (APA5) and a viability dye (all from BioLegend) in staining buffer (PBS containing 1% FCS) and kept on ice and in the dark for 30 min. The cells were washed twice with 2 ml staining buffer and fixed with 200 μl fixation buffer (eBioScience; San Diego, CA, USA) before analysis by flow cytometry. All antibodies were titrated using mouse splenocytes at different dilutions with the final dilution applied found to be most appropriate for the particular batch of antibody used and our flow cytometer set up.
For intracellular staining, the single-cell suspension was treated with Perm/Fix buffer (eBioscience) followed by pre-titrated monoclonal antibodies conjugated with different fluorochromes to FoxP3 (FJK-16S, eBioscience) or FluoZin-3-AM (ThermoFisher). After 30 min incubation on ice or at room temperature, the cells were washed twice with 2 ml staining buffer and analysed by flow cytometry. FoxP3 was titrated using mouse splenocytes at different dilutions with the final dilution applied found to be appropriate for the batch used and our flow cytometer set up. For Fluozin-3-AM, mouse islets were used to titrate the antibody, with 1:2000 dilution used found to be appropriate for the particular batch of antibody used and our flow cytometer set up. Dilutions were determined where they gave the clearest separation from the negative background or isotype control.
Insulin release assay
An insulin release assay was performed as previously described  with modification. Hand-picked pancreatic islets from randomly selected Tlr9−/− and Tlr9+/+ NOD or C57BL/6 mice (5–6 weeks old) were equally distributed to 30 islets/tube after stabilising with low-glucose KRB buffer. The islets were then stimulated with KRB containing high glucose (25 mmol/l) and the supernatant fractions were harvested every 5 min after glucose stimulation. Secreted insulin in the supernatant fractions was measured using the insulin RIA kit (EMD-Millipore, Burlington, ME, USA).
Evaluation of islet mass
Ex vivo pancreases from randomly selected 5–6-week-old female Tlr9−/− NOD and Tlr9+/+ NOD mice were fixed in periodate–lysine–paraformaldehyde, sucrose infused and then frozen in Tissue-Tek OCT (Bayer, Elkhart, IN, USA). The pancreas was cut in its entirety into hundreds of 10 μm thick sections and every tenth section was stained with haematoxylin alone (to better visualise the islets) and photographed under the microscope. Islet mass was measured using Image J software (NIH, Bethesda, MD, USA). H&E staining of sections was conducted purely for improving the contrast of the images for the photographs presented in Fig. 4b.
In vitro TLR9 antagonist treatment
Freshly isolated islets from Tlr9+/+ NOD mice (5-week-old females) were cultured overnight with the TLR9 antagonist CpG- oligodeoxynucleotides (ODN) (2088; Invivogen, San Diego, CA, USA) or control CpG-ODN (Invivogen), both at 10 μg/ml. After extensive washing, a single-cell suspension was prepared as described earlier and stained with fluorochrome-conjugated monoclonal antibodies to CD45, CD140a and FluoZin-3-AM before analysis by flow cytometry. Another set of freshly isolated islets from Tlr9+/+ NOD mice was used for insulin release assay, after overnight culture in the presence of the TLR9 antagonist CpG-ODN or control CpG-ODN.
In vivo treatment with TLR9 antagonist or chloroquine and diabetes development
Randomly chosen Tlr9+/+ female NOD mice were treated with TLR9 antagonist CpG-ODN (2088) or control ODN, 10 μg/mouse, administered as two i.p. injections, 3 days apart, 1 week after mating. Another set of randomly chosen Tlr9+/+ pregnant female NOD mice were treated with chloroquine (20 μg/g body weight), administered as two i.p. injections, 3 days apart. The female offspring from the treated mothers were investigated for CD140a-expressing islet beta cells, the number of islet beta cells and insulin-secreting function at ~5 weeks old. A third group of randomly chosen pregnant female Tlr9+/+ NOD mice were also treated with antagonist CpG-ODN or control ODN and the natural history of diabetes development was observed in the female progeny of the treated pregnant mice.
No data were excluded and all viable mice within the different genotypes were included, with the exception of any obvious runts or under-developed mice. No outcomes or conditions were measured or used that are not reported in the results section. Statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA). Diabetes incidence was compared using logrank test. The in vivo and in vitro assays were analysed with Student’s unpaired t test or ANOVA for statistical significance.
TLR9 deficiency suppressed type 1 diabetes development and enhanced islet beta cell function
Increased islet and beta cell number in the absence of TLR9 in NOD mice
Increased islet beta cells expressing CD140a in the absence of TLR9 in NOD mice
Treatment with TLR9 antagonist resulted in increased CD140a-expressing islet beta cells and number of beta cells, improved beta cell function and protected Tlr9 +/+ NOD mice from diabetes development
In this study, we have identified a novel function of TLR9, quite distinct from its role in innate immunity. We showed that TLR9-deficient mice have more pancreatic islets and, correspondingly, more islet beta cells, with increased glucose-stimulated insulin secretion in vitro and improved glucose tolerance in vivo. This was not due to increased resistance to beta cell death. Rather, we found increased expression of genes encoding PDX-1 and NGN3, transcription factors associated with beta cell development, suggesting that the increase in beta cell mass was related to promotion of beta cell growth. Although many growth factors regulate islet beta cell development [28, 32], in linking TLR9 deficiency and islet beta cell development, we found that the proportion of islet beta cells expressing CD140a was increased in TLR9-deficient mice. Confirming that this effect was associated with TLR9 deficiency, we showed, using TLR9 antagonism, that inhibition of the TLR9 signalling pathway in islets from TLR9-sufficient mice led to an increased number of CD140a-expressing beta cells and enhanced insulin secretion in response to glucose stimulation. Our results thus demonstrate a novel link between TLR9 and CD140a, a growth factor that has been reported to regulate islet beta cell proliferation .
Islet beta cells display distinct phases of significant growth in the fetal and neonatal periods, whereas there is little increase in islet beta cell numbers in adulthood in either mice or humans [33, 34]. Proliferation and survival are among the functions promoted by platelet-derived growth factor (PDGF) signalling through the PDGF receptors, of which CD140a is one of two main receptor isoforms for PDGF. This is a receptor tyrosine kinase and it is expressed in cells of mesenchymal origin, including the pancreas . It has been suggested that the human CD140a promoter has a binding site for c-Rel , which is a subunit of the NFκB protein complex and plays an important role in development, immunity and diseases, including type 1 diabetes [37, 38, 39]. Expression of CD140a is normally age-dependent in mouse pancreatic islet beta cells, reaching a peak at around the age of 2 weeks and declining once mice reach adulthood . If this receptor is lost prematurely, as shown by gene mutation experiments, beta cell proliferation and expansion are impaired . Our current results suggest that TLR9 may be one of the factors involved in the control of the age-dependent expression of CD140a, and may negatively regulate the expression of CD140a. Blocking TLR9 signalling, either by genetic targeting or by inhibition via small molecules early in life, results in an increase in CD140a expression and a corresponding increase in islet beta cells later in life.
There is increasing evidence to suggest that TLRs not only recognise exogenous ligands from microbes but also recognise endogenous ligands from both normal and damaged cells. As TLR9 recognises CpG motifs, present in bacterial DNA or endogenous DNA, that are released from both physiological and pathological dying cells , it could play a role in tissue remodelling. Although the gut microbiota differs in composition when comparing Tlr9−/− NOD and Tlr9+/+ NOD mice, protection from diabetes and enhanced beta cell development and function were not associated with this difference (data not shown). It is particularly interesting that TLR9 is linked to expression of a growth factor receptor, signalling through which affects growth and development in a tissue-specific manner. Islet beta cells undergo significant growth and remodelling during prenatal development  but the capacity for neogenesis and regeneration of beta cells is lost later in life. Marked beta cell hyperplasia occurs during neonatal development and the role of CD140a in beta cell proliferation is age-dependent . In our experiments, brief treatment of pregnant Tlr9+/+ NOD mice with a TLR9 antagonist oligonucleotide, as well as with chloroquine (which also antagonises TLR9), significantly enhanced beta cell growth. This was accompanied by enhanced insulin secretion in response to glucose stimulation, as the mice developed into adulthood, and also coincided with an increased percentage of CD140a-expressing beta cells, suggesting the association of the two processes. We did not examine the effect of chloroquine on islet beta cells directly in vitro, as we think it is important to study the drug effect in vivo; although chloroquine is not specific for islet beta cells, we did not observe any noticeable systemic adverse effects.
TLR signalling in mammals has been mainly linked to innate immunity. Our results suggest a novel effect of TLR9 on growth and development in addition to its role in innate immunity. The fact that this brief inhibition of TLR9 signalling, early in life, led to protection from autoimmune diabetes development in Tlr9+/+ NOD mice, similar to the phenotype seen in Tlr9−/− NOD mice, suggests a possible means of improving islet reserve. The inhibition of the development of diabetes is likely to be a combination of increased beta cell capacity (referring to increase in number of beta cells, improved sensitivity to glucose and increased insulin production), together with the immunological changes that we, and others, have previously reported [9, 10]. These changes include increased expression of CD73 and reduced production of proinflammatory cytokines , together with a reduction in activation of autoreactive diabetogenic CD8 T cells, all of which occur as a result of TLR9 inhibition . Inhibition of TLR9 has not been explored in human type 1 diabetes; however, pre-clinical tests for identifying an effective and safe dose, route and timing of any potential agent would need to be conducted first.
We conclude that TLR9 negatively regulates the development of pancreatic islets and insulin-secreting beta cells, mediated, at least in part, by CD140a. Our findings provide novel insight into the function of TLR9 beyond the immune cells and also suggest a new direction for the design of preventive and/or therapeutic strategies for diabetes.
We thank X. Zhang (Internal Medicine, Yale University, USA) for taking excellent care of the animals used in this study.
ML, JP, NT, CH and JG performed experiments. ML, JP, JAP, FSW and LW analysed the data and LH and HZ carried out bioinformatic analysis. LW and FSW wrote the manuscript and JAP edited the manuscript. All authors reviewed and approved the manuscript. LW initiated and designed the study and is the guarantor of the work.
This work was funded by the National Institutes of Health (grant numbers: DK-092882, DK-100500 and Mouse Genetic Core of P30 DK-405735).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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