Interferon-α promotes neo-antigen formation and preferential HLA-B-restricted antigen presentation in pancreatic ß-cells.

Interferon (IFN)-α is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic ß-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed ß-cells. IFN-α increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting ß-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN-α only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and we identified islet-infiltrating CD8+ T-cells from T1D donors reactive to HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates TNF-α-induced human beta cell dysfunction and death.

AIMS/HYPOTHESIS: TNF-α plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-α inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-α, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. METHODS: EndoC-ßH1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-α (2000 U/ml) or TNF-α (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. RESULTS: PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-α and/or TNF-α by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-α, TNF-α or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-α-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-α cytotoxicity. CONCLUSIONS/INTERPRETATION: We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-α in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-α.

ADAR1-dependent editing regulates human ß cell transcriptome diversity during inflammation.

Introduction: Enterovirus infection has long been suspected as a possible trigger for type 1 diabetes. Upon infection, viral double-stranded RNA (dsRNA) is recognized by membrane and cytosolic sensors that orchestrate type I interferon signaling and the recruitment of innate immune cells to the pancreatic islets. In this context, adenosine deaminase acting on RNA 1 (ADAR1) editing plays an important role in dampening the immune response by inducing adenosine mispairing, destabilizing the RNA duplexes and thus preventing excessive immune activation. Methods: Using high-throughput RNA sequencing data from human islets and EndoC-ßH1 cells exposed to IFNα or IFNγ/IL1ß, we evaluated the role of ADAR1 in human pancreatic ß cells and determined the impact of the type 1 diabetes pathophysiological environment on ADAR1-dependent RNA editing. Results: We show that both IFNα and IFNγ/IL1ß stimulation promote ADAR1 expression and increase the A-to-I RNA editing of Alu-Containing mRNAs in EndoC-ßH1 cells as well as in primary human islets. Discussion: We demonstrate that ADAR1 overexpression inhibits type I interferon response signaling, while ADAR1 silencing potentiates IFNα effects. In addition, ADAR1 overexpression triggers the generation of alternatively spliced mRNAs, highlighting a novel role for ADAR1 as a regulator of the ß cell transcriptome under inflammatory conditions.

Pro-Inflammatory Cytokines Promote the Transcription of Circular RNAs in Human Pancreatic ß Cells.

Circular RNAs (circRNAs) have recently been implicated in impaired ß-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-ßH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human ß cells. We identified ~5000 ß-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for ß-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as 'sponges' or 'decoys'. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated ß-cell destruction in type 1 diabetes.

Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic ß cells.

IFNα is a key regulator of the dialogue between pancreatic ß cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human ß cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-ßH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of ß cell neoantigens, suggesting that it is a central player of the effects of IFNα on ß cells that contribute to trigger and amplify autoimmunity in T1D.

A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes.

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis.

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-ßH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.We identified a total of 264 genes stably expressed in EndoC-ßH1 cells and human islets following cytokines - or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

The RNA-binding profile of the splicing factor SRSF6 in immortalized human pancreatic ß-cells.

In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.

Persistent or Transient Human ß Cell Dysfunction Induced by Metabolic Stress: Specific Signatures and Shared Gene Expression with Type 2 Diabetes.

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.

SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic ß-Cells and in the Human Pancreas Microvasculature.

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing ß-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human ß-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the ß-cell line EndoC-ßH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct ß-cell virus tropism.

Molecular Footprints of the Immune Assault on Pancreatic Beta Cells in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic disease caused by the selective destruction of the insulin-producing pancreatic beta cells by infiltrating immune cells. We presently evaluated the transcriptomic signature observed in beta cells in early T1D and compared it with the signatures observed following in vitro exposure of human islets to inflammatory or metabolic stresses, with the aim of identifying "footprints" of the immune assault in the target beta cells. We detected similarities between the beta cell signatures induced by cytokines present at different moments of the disease, i.e., interferon-α (early disease) and interleukin-1ß plus interferon-γ (later stages) and the beta cells from T1D patients, identifying biological process and signaling pathways activated during early and late stages of the disease. Among the first responses triggered on beta cells was an enrichment in antiviral responses, pattern recognition receptors activation, protein modification and MHC class I antigen presentation. During putative later stages of insulitis the processes were dominated by T-cell recruitment and activation and attempts of beta cells to defend themselves through the activation of anti-inflammatory pathways (i.e., IL10, IL4/13) and immune check-point proteins (i.e., PDL1 and HLA-E). Finally, we mined the beta cell signature in islets from T1D patients using the Connectivity Map, a large database of chemical compounds/drugs, and identified interesting candidates to potentially revert the effects of insulitis on beta cells.

Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice.

The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.

An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells.

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.

The T1D-associated lncRNA Lnc13 modulates human pancreatic ß cell inflammation by allele-specific stabilization of STAT1 mRNA.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13 Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic ß-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in ß-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

The impact of proinflammatory cytokines on the ß-cell regulatory landscape provides insights into the genetics of type 1 diabetes.

The early stages of type 1 diabetes (T1D) are characterized by local autoimmune inflammation and progressive loss of insulin-producing pancreatic ß cells. Here we show that exposure to proinflammatory cytokines reveals a marked plasticity of the ß-cell regulatory landscape. We expand the repertoire of human islet regulatory elements by mapping stimulus-responsive enhancers linked to changes in the ß-cell transcriptome, proteome and three-dimensional chromatin structure. Our data indicate that the ß-cell response to cytokines is mediated by the induction of new regulatory regions as well as the activation of primed regulatory elements prebound by islet-specific transcription factors. We find that T1D-associated loci are enriched with newly mapped cis-regulatory regions and identify T1D-associated variants disrupting cytokine-responsive enhancer activity in human ß cells. Our study illustrates how ß cells respond to a proinflammatory environment and implicate a role for stimulus response islet enhancers in T1D.

Coxsackievirus B Tailors the Unfolded Protein Response to Favour Viral Amplification in Pancreatic ß Cells.

Type 1 diabetes (T1D) is an autoimmune disease characterized by islet inflammation and progressive pancreatic ß cell destruction. The disease is triggered by a combination of genetic and environmental factors, but the mechanisms leading to the triggering of early innate and late adaptive immunity and consequent progressive pancreatic ß cell death remain unclear. The insulin-producing ß cells are active secretory cells and are thus particularly sensitive to endoplasmic reticulum (ER) stress. ER stress plays an important role in the pathologic pathway leading to autoimmunity, islet inflammation, and ß cell death. We show here that group B coxsackievirus (CVB) infection, a putative causative factor for T1D, induces a partial ER stress in rat and human ß cells. The activation of the PERK/ATF4/CHOP branch is blunted while the IRE1α branch leads to increased spliced XBP1 expression and c-Jun N-terminal kinase (JNK) activation. Interestingly, JNK1 activation is essential for CVB amplification in both human and rat ß cells. Furthermore, a chemically induced ER stress preceding viral infection increases viral replication, in a process dependent on IRE1α activation. Our findings show that CVB tailors the unfolded protein response in ß cells to support their replication, preferentially triggering the pro-viral IRE1α/XBP1s/JNK1 pathway while blocking the pro-apoptotic PERK/ATF4/CHOP pathway.

PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.

BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.

When one becomes many-Alternative splicing in ß-cell function and failure.

Pancreatic ß-cell dysfunction and death are determinant events in type 1 diabetes (T1D), but the molecular mechanisms behind ß-cell fate remain poorly understood. Alternative splicing is a post-transcriptional mechanism by which a single gene generates different mRNA and protein isoforms, expanding the transcriptome complexity and enhancing protein diversity. Neuron-specific and certain serine/arginine-rich RNA binding proteins (RBP) are enriched in ß-cells, playing crucial roles in the regulation of insulin secretion and ß-cell survival. Moreover, alternative exon networks, regulated by inflammation or diabetes susceptibility genes, control key pathways and processes for the correct function and survival of ß-cells. The challenge ahead of us is to understand the precise role of alternative splicing regulators and splice variants on ß-cell function, dysfunction and death and develop tools to modulate it.