Effects of Exogenous Interleukin-18 on Islet Function and Immune Mechanisms in Type 1 Diabetic Mice

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However, the effect of exogenous IL-18 and immune mechanisms involved have not yet been fully elucidated. This study aimed to inquire into effects of exogenous IL-18 and immune mechanisms in T1DM. Methods : A type 1 diabetes mouse model was established using a single intraperitoneal injection of freshly prepared STZ 180 mg per body weight. All the mice were all separated into three groups: diabetic mice treated with recombinant IL-18 (1 ug/mouse) every other day (D + IL-18 group) ; diabetic mice treated with phosphate-buffered saline (D group); and a control group with non-diabetic mice receiving no treatment. Fasting blood glucose levels were measured throughout the treatment period. At the end of the 10 days treatment, an intraperitoneal glucose tolerance test was performed. To assess islet morphology and apoptosis, hematoxylin-eosin staining, immunohistochemistry, and immunofluorescence staining(insulin and elevate caspase 3 co-localization) were conducted. Flow cytometry was used to explore immune mechanisms of islet injury. Results : The type 1 diabetes mellitus model was successfully established. During the first 7 days of recombinant IL-18 treatment, the D + IL-18 group’s mice showed significantly lower fasting blood glucose levels than the D group’s mice (p < 0.05); however, glucose levels increased thereafter (p < 0.05). The intraperitoneal glucose tolerance test showed a larger area under the curve in the D + IL-18 group than in both the D and control groups (p < 0.05), indicating impaired glucose tolerance. Histological analysis revealed disrupted islet architecture and increased elevate caspase 3 expression, consistent with islet dysfunction. Flow cytometry demonstrated elevated proportions of CD11b⁺F4/80⁺ macrophages and CD3⁻NK1.1⁺ natural killer cells in the pancreas of IL-18–treated mice (p < 0.05). Conclusion : Exogenous IL-18 temporarily ameliorated fasting blood glucose in T1DM mice, followed by deterioration in fasting blood glucose and islets function after day 7 ,potentially due to enhanced infiltration of CD11b⁺F4/80⁺ macrophages and CD3⁻NK1.1⁺ natural killer cells. exogenous interleukin-18 islet function immune mechanisms T1DM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Pediatric diabetes is an endocrine disorder that poses a significant threat to the health of children, with Type 1 Diabetes Mellitus (T1DM) being the predominant form [1]. In China, the prevalence of T1DM among children is estimated to range from 2 to 5 cases per 100,000 individuals, with an increasing trend toward earlier onset [2][3]. In 2022, the number of people living with T1D was estimated to be approximately 8.75 million worldwide, with 650,000 new cases that year. [4] The underlying cause of T1DM is damage of islets β-cells, causing severely impaired insulin secretion. Xian Z et al. [5] demonstrated through extensive animal studies that IL-18 plays a critical role in regulating insulin secretion and glucose metabolism. IL-18 acts through two key receptors: the IL-18 receptor (IL-18R) and the Na⁺/Cl⁻ cotransporter (NCC). Specifically, IL-18 is predominantly expressed in pancreatic α-cells, NCC in β-cells, and IL-18R in acinar cells. Deficiency in these receptors has been shown to reduce islet size, impair β-cell proliferation, and decrease insulin secretion. Moreover, under conditions such as diet-induced glucose intolerance or streptozotocin (STZ)-induced hyperglycemia, receptor deficiency exacerbates β-cell apoptosis. Also,the effect of interleukin-18 on pancreatic islet function is closely related to immune cells.Xian Z et al [5] considered acinar cell IL18r mediates IL18 activities inhibited macrophages expansion and inflammation in the hyperglycemic pancreas.These results indicate that endogenous IL-18 plays a critical protective role in maintaining islet homeostasis and immune mechanisms are involved. Conversely, Oikawa et al. [6] found that IL-18-expressing plasmid DNA increased the infiltration of CD4⁺ T cells and macrophages in the pancreas. Similarly, Li etal [7] identified immune cell infiltration, particularly CD4⁺ T cells and macrophages, as a central mechanism in T1DM pathogenesis in mouse models. These findings deduced exogenous IL-18 may could contribute to islet damage through pro-inflammatory pathways, while the effects and mechanisms of exogenous rIL18 on pancreatic function were not verified in live mice. To explore the differing effects of endogenous and exogenous IL-18, our study investigates the impact of exogenously administered IL-18 on islet function and immune responses in a mouse model of type 1 diabetes. We aim to elucidate the immune mechanisms underlying islet injury and dysfunction, with particular attention to the role of IL-18 in modulating immune cell infiltration. Additionally, we seek to clarify how different treatment course and cumulative dose of rIL-18 affect pancreatic islets, thereby providing a theoretical basis for developing IL-18-targeted therapeutic strategies for childhood diabetes. 2. Materials and Methods 2.1 Animals Male C57BL/6 mice (18–20 g, 5 weeks old) were obtained from Hangzhou Ziyuan Experimental Animal Technology Co., LTD. The mice were housed individually in a controlled environment, which was regulated at 25 ± 2℃ and 50 ± 5% relative humidity, with a 12-h light-dark cycle programmed from 08:00 to 20:00. Following one-week acclimatization period, 21 mice were randomly assigned into three groups: IL-18-treated diabetic group (D + IL-18; n = 7);diabetic group (D; n = 7), non-diabetic control group (CON; n = 4).All animal studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of Anhui Provincial Hospital, affiliated with the First Affiliated Hospital of the University of Science and Technology of China.. (Approval No. 2025-N(A)-0115). All procedures were performed in strict compliance with the Guide for the Care and Use of Laboratory Animals. We induced type 1 diabetes mellitus (T1DM) in 6-week-old male mice with a single intraperitoneal injection of freshly prepared STZ (180 mg/kg body weight) (Solarbio, China) dissolved in citrate buffer (pH 4.5), following overnight fasting. Blood glucose levels were measured after 4 weeks using a OneTouch II glucometer (Lifescan, Inc.) to confirm diabetic status (defined as fasting blood glucose [FBG] > 16.6 mmol/L). After four weeks (when mice were 10 weeks old), diabetic mice were randomly divided into two subgroups: one received intraperitoneal injections of rmIL-18 (1 ug/mouse) (Medical & Biological Laboratories, Japan)every other day for 10 days, and the other received phosphate-buffered saline (PBS). Non-diabetic control mice received no treatment. 2.2 Intraperitoneal glucose tolerance test (IPGTT) After a 12-hour fast(8AM-8PM), mice were administered glucose intraperitoneally (1 g/kg) ༈8PM༉. Blood glucose was then determined at intervals of 0 (baseline), 30, 60, and 120 minutes using Luo's glucose meter. Subsequently, the total area under the curve(AUC) for glucose was calculated according to the trapezoidal rule. 2.3 Organ harvesting The pancreas were harvested after IPGTT, rinsed with PBS, and processed as follows: pancreatic portions were snap-frozen and stored at − 80℃ for further analysis; other portions were fixed in 10% formaldehyde for paraffin embedding and histological staining; remaining pancreatic tissue were processed into single-cell suspensions for flow cytometry analysis. 2.4 Hematoxylin-eosin (HE) staining Paraffin-embedded pancreatic tissue were sectioned at 5 µm, dewaxed with xylene, rehydrated in graded alcohols, and stained with 0.1% hematoxylin (G1120, Solarbio, Beijing, China) for 1 minute, followed by 0.5% eosin (G1120, Solarbio, Beijing, China) for 3 minutes, both at room temperature. Sections were mounted with glycerogelatin and imaged using an optical microscope (Axio Scope AI, Zeiss, Germany). Pancreatic morphology was observed and examined. 2.5 Immunohischemistry (IHC) staining Paraffin sections (5 µm) were cut into thick sections, which were processed through xylene dewaxing, graded alcohol rehydration, and antigen retrieval by heating in pH 6.0 citrate buffer (G1201, Servicebio, Wuhan, China) at 95℃ for 30 minutes. After cooling to room temperature, endogenous peroxidase activity was blocked using hydrogen peroxide. Sections were incubated overnight at 4°C with anti-insulin rabbit polyclonal antibody (pAB) (bsm-60010R, Bioss, Beijing, China), followed by a secondary antibody (G1302, Servicebio, Wuhan, China) for 60 minutes. After counterstaining with 0.1% hematoxylin (G1120, Solarbio, Beijing, China), slides were imaged using an optical microscope (Axio Scope AI, Zeiss, Germany). The proportion of insulin-positive cells within the islets were analyzed. 2.6 Immunofluorescence (IF) staining Paraffin sections (5 µm) were cut into thick sections, which were processed through xylene dewaxing, graded alcohol rehydration, and antigen retrieval by heating in pH 6.0 citrate buffer (G1201, Servicebio, Wuhan, China) to 95℃ and then immersed for 30 minutes and cooled to room temperature. Sections were incubated with anti-insulin mouse monoclonal antibody (mAb) (GB13121, Servicebio, Wuhan, China) and Anti-Cleaved-Caspase-3 rabbit pAb (GB11532, Servicebio, Wuhan, China) primary antibodies overnight at 4°C, then incubated with secondary antibodies (GB21303, GB22301, Servicebio, Wuhan, China) for 90 minutes. Cell nuclei were counterstained with DAPI (G1012, Servicebio, Wuhan, China), and slides were mounted with an anti-fluorescence quenching sealer (G1401, Servicebio, Wuhan, China). Fluorescence images were acquired with Zeiss Axio Imager M2microscope(Axio Imager M2,Zeiss,Germany).The proportion and intensity of insulin and cleaved caspase-3 expression were quantified. 2.7 Flow cytometry (FC) analysis Fresh spleen and pancreatic tissues were enzymatically digested using collagenase IV (BS165, Bioshap, Jiangsu, China) in RPMI-1640 medium (PM150110C, Wuhan, China) and passed through 40 µm filters. Cells suspensions were centrifuged at 600 g for 10 minutes, and red blood cells were lysed using RBC lysis buffer (R1010, Solarbio, Beijing, China). After washing with PBS, 100 µL of cell suspension was stained with the following antibodies: Fixable Viability Dye eFluor™ 506 (65-0866-14, Thermo Fisher, USA), anti-CD45-Pacific blue (Proteintech), anti-CD3-PE (Proteintech), anti-CD4-APC (Proteintech), anti-NK1.1-FITC (Proteintech), anti-CD11b-Apc-cy7 (Proteintech), and anti-F4/80-PE-Texas Red (Proteintech). Samples were incubated on ice for 30 minutes in the dark, then analyzed using a BD Fortessa flow cytometer. Data were processed with FACSDIVA software (BD Biosciences). 2.8 Statistical Analysis FBG levels and immune cell distributions were analyzed using one-way analysis of variance (ANOVA). Non-parametric data, including insulin and cleaved caspase-3 expression levels were analyzed using the Kruskal-Wallis test. All statistical analyses were performed with GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. 3. Results 3.1 Exogenous rmIL-18 temporarily ameliorates fasting blood glucose (FBG) in T1DM mice, followed by deterioration after day 7. Experimental T1DM models were successfully established:at day 0,we started modeling ,at day 28, FBG levels exceeded 16.7 mmol/L in both the D and D + IL-18 groups.At day 29, the mice of D + IL-18 groups received rmIL18(1ug/d)every other day for10 days,as shown in Fig. 1 A, and the experimental design is illustrated in Fig. 1 B: the mice of D + IL-18 groups received rmIL18; D groups received PBS;Con groups receibed no treatment. FBG and body weight were monitored across all groups. Compared to the CON group, the body weights of the D and D + IL-18 groups were significantly decreased (Fig. 1C1), while FBG levels were significantly elevated (Fig. 1C2). Treatment began at the start of week 10. On days 1, 3, and 5 of treatment, the FBG levels in the D + IL-18 group were significantly lower than those in the untreated diabetic group (D) (p 0.05),as depicted in Figs. 2 D. On day 9, a marked increase in FBG was observed in the D + IL-18 group compared with the D group (p < 0.05), as depicted in Figs. 2 E. By the end of treatment, No statistically significant difference in body weight as discernible between the D and D + IL-18 groups, as depicted in Figs. 2 F.Overall, FBG levels in the D + IL-18 group initially decreased and then gradually increased, as depicted in Fig. 3 A.in the D + IL-18 group,FBG levels showed an initial decline, followed by a gradual increase over time. 3.2 Exogenous rmIL-18 impairs islet function after day 7 of treatment Before the IPGTT, mice were fasted for 12 hours. The decrease in FBG was more rapid in the D group than in the D + IL-18 group (Fig. 3 B). In the IPGTT results (Fig. 3 C), the fasting (0 minutes) glucose level in the D + IL-18 group was significantly higher than in the D group. At all subsequent time points (15, 30, 60, 90, and 120 minutes), blood glucose remained higher in the D + IL-18 group. The AUC for glucose was significantly increased in the D + IL-18 group compared to both the D and CON groups (p < 0.05). Additionally, AUC was significantly lower in the CON group than in the D group (p < 0.05) (Fig. 3 C). To a certain extent, the findings revealed that the rIL-18 treated group exhibited a more pronounced impairment in islet function compared to the diabetic group. 3.2.2 HE staining Histological analysis are shown in Figs. 4A–C. In the CON group, islet morphology was well-preserved with clearly visible cell boundaries and minimal signs of denaturation or necrosis. In contrast, islets in both the D and D + IL-18 groups appeared irregular and structurally disrupted. The D + IL-18 group exhibited more pronounced cellular sparsity and vacuolation than the D group. A more severe disruption of the islet architecture was observed in diabetic mice following 7 days of rIL-18 treatment. 3.2.3 IHC staining As shown in Figs. 4D–F, islet size and β-cell proportion differed among groups. The control group exhibited the highest β-cell ratio and mean insulin density. These measures were reduced in the D group and further decreased in the D + IL-18 group, as quantified in Figs. 4G–H. The already impaired insulin secretion in diabetic mice was further aggravated by a 7-day rIL-18 treatment. 3.2.4 IF staining To evaluate apoptosis in pancreatic islets, co-staining for insulin (green), cleaved caspase-3 (red), and DAPI (blue) was performed (Figs. 5 A –L). The D + IL-18 group showed a higher proportion and intensity of cleaved caspase-3-positive cells compared to the D group. Both diabetic groups had significantly more caspase-3 expression than the CON group, as shown in Figs. 5 M –N. Apoptosis of pancreatic islets was markedly enhanced in diabetic mice following a 7-day course of interleukin 18. 3.3 Exogenous rmIL-18 promotes immune cell infiltration in the pancreas after day 7 To investigate the immune mechanism of islet dysfunction following rmIL-18 treatment, we analyzed immune cell infiltration in the pancreas. Flow cytometry assessed the proportions of T cells, NK cells, macrophages, and CD45 + CD11b + cells. In the pancreas (Figs. 6A–D), the proportions of CD45 + CD11b + cells and NK1.1 cells were significantly elevated in the D + IL-18 group compared to both the D and CON groups (p < 0.05) (Fig. 7 A–B). No significant differences were found between the D and CON groups (Figs. 7 A–B). For CD3 + CD4 + T cells in the pancreas, the proportion was significantly lower in the D + IL-18 group compared to both the D and CON groups (p < 0.05) (Fig. 7 A–B). Thus, our findings demonstrate that rIL-18 drives the infiltration of macrophages and NK cells into the pancreatic tissue.. 4. Discussion IL-18 is a member of the IL-1 cytokine family that plays a centeral role in activating both innate and adaptive immunity [8]. It acts on Th1 cells, NK cells, B cells, and macrophages [9], and is implicated in the pathogenesis of multiple diseases [10]. Elevated IL-18 levels have been reported in conditions such as systemic lupus erythematosus [11], hypertension, chronic kidney disease [12], and type 2 diabetes and obesity [13]. However, research on the role of IL-18 in T1DM remains limited. Xian Z etal[5].demonstrated.endogenous IL-18 plays a critical protective role in maintaining islet homeostasis and macrophage were involved. no published studies have systematically investigated the effects of exogenous rmIL-18 on islet function and immune mechanisms in diabetic mice.Oikawa et al. [6] demonstrated that plasmid DNA expressing IL-18 accelerated the onset of diabetes in 4-week-old NOD mice, though the mechanism related to islet function and immune responses were not examined. Conversely, Rothe et al. [14] reported that exogenous IL-18 administration could prevent diabetes onset under certain conditions. Our findings showed that short-term rmIL-18 treatment led to a transient improvement in glycemic control during the first 7 days. This effect may be attributed to IL-18 acting on NCC in pancreatic islets, enhancing insulin gene expression, inhibiting macrophage infiltration, and reducing local inflammation, consistent with the findings of Zhang et al. [5]. Our study confirmed that rmIL-18 led to elevated FBG levels after 7 days of treatment, as evidenced by phenotypic analysis in diabetic mice. During a 12-hour fast before IPGTT, the rmIL-18-treated group declined significantly less than those in the untreated D group.Since the rate and extent of glucose reduction during fasting are closely related to pancreatic function[15], this observation supports the conclusion that islet function deteriorated following rmIL-18 administration.these findings was consistent with prior studies, such as by Frigerio et al. [16], who reported that IL-18 promotes islet cell death through pro-inflammatory cytokines and immune cell infiltration. Similarly, Dao et al. [17] demonstrated that IL-18 activates NK cells, which then interact with islet cells and induce programmed cell death. The pathogenesis of T1DM is primarily driven by the infiltration and attack of T cells and other immune cells on pancreatic β-cells, ultimately leading to their destruction and the development of an organ-specific autoimmune disease characterized by insulin deficiency. [18] Various immune cells, including T cells, B cells, NK cells, macrophages, and group 2 innate lymphoid cells (ILC2), have been shown to infiltrate pancreatic islets [19], where they interact with β-cells and promote their apoptosis. IL-18, a pro-inflammatory cytokine, plays key role in this process by acting on Th1 cells, NK cells, B cells, and macrophages [9], thereby modulating immune responses and contributing to target organ damage. In our study, flow cytometry revealed that IL-18 treatment after 7days significantly increased the proportion of pancreatic macrophages and NK cells, while T cells were markedly reduced, potentially due to the expansion of macrophages and NK cells. These immune changes, together with the observed phenotypic deterioration in islet structure and function following IL-18 treatment 7 days, support the conclusion that the decline in islet function is closely related to increased macrophage and NK cell infiltration and their cytotoxic interactions with pancreatic β-cells, which was consistent with the Li C[7] and Sophie L Walker[19]. In summary, the effects of rmIL-18 on T1DM mice are complex and multifaceted. On one hand, IL-18 may enhance insulin synthesis by acting on the NCC receptor in pancreatic islets, thereby promoting insulin gene expression, inhibiting macrophage infiltration[4], in the early treatment courses and with smaller cumulative doses, rIL18 had similar effects to endogenous interleukin-18 in diabetic mice. On the other hand, as the treatment course and cumulative dose increased, rmIL-18 also facilitates the infiltration of immune cells, particularly macrophages and NK cells, into the pancreatic islets via IL-18R signaling, contributing to insulitis and accelerating islet apoptosis. The overall impact of exogenous IL-18 on islet function appears to depend on multiple factors, including treatment duration and cumulative dose, which together determine whether its net effect is protective or detrimental. Limitation: Due to there was no precedent for exogenous interleukin 18 treatment of diabetic mice, we were unable to conduct IPGTT, collect tissue samples, or investigate the underlying mechanisms during the early phase of blood glucose reduction. As a result, the potential protective effects of exogenous IL-18 at the initial stage of treatment could not be fully explored. Further studies are needed to address this gap and further clarify the early immunometabolic responses to IL-18. 5. Conclusion This study investigated the effects of exogenous rmIL-18 on islet function in a mouse model of T1DM. During the first 7 days of treatment, FBG levels steadily declined. After 7 days of treatment, FBG levels began to rise again, and IPGTT revealed a deterioration in islet function. Histological analysis via HE and IHC staining showed more severe islet damage in the rmIL-18-treated group compared to the PBS-treated diabetic group, while IF confirmed increased islet cell apoptosis. Flow cytometry suggested that this deterioration may be associated with increased infiltration of macrophages and NK cells into the pancreas following rmIL-18 treatment. Declarations AUTHORS’ CONTRIBUTIONS Experimental design was proposed by Xian Zhang and Shenggang Ding,all experiments were guided by Xian Zhang.Experiment completed,data acquisition, data analysis, and interpretation were carried out by Mei Xiong.The experimental data were processed by Li Ting ,all of them provided the final approval of the version to be published. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study was approved by the Ethics Committee of Anhui Provincial Hospital affiliated to the First Affiliated Hospital of University of Science and Technology of China and use Committee­【2025-N(A)-0115】. FUNDING This research was supported by the The National Natural Science Fund（52273113） CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. 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PMID: 9725214 Zheng CA,Fu JF(2020) Pathogenesis of type 1 diabetes. Int J Pediatrics 47(4),274-278 Walker SL, Leete P , Boldison J(2025)Tissue Resident and Infiltrating Immune Cells: Their Influence on the Demise of Beta Cells in Type 1 Diabetes. Biomolecules 15,3,441?DOI: 10.3390/biom15030441;PMID: 40149976;PMCID: PMC11939886 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 22 Jan, 2026 Reviewers agreed at journal 22 Dec, 2025 Reviewers invited by journal 22 Dec, 2025 Editor invited by journal 28 Nov, 2025 Editor assigned by journal 25 Nov, 2025 First submitted to journal 25 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8181438","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":564118876,"identity":"2f3ec774-c2e5-4987-b0fd-fe35128593b7","order_by":0,"name":"mei 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1","display":"","copyAsset":false,"role":"figure","size":129431,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design and data of mice before the experiment\u003c/p\u003e\n\u003cp\u003eA Successful establishment of experimental T1DM mouse models\u003c/p\u003e\n\u003cp\u003eB Schematic overview of the experimental procedures for each group\u003c/p\u003e\n\u003cp\u003eC1 Body weight of the three groups at week 10, prior to treatment\u003c/p\u003e\n\u003cp\u003eC2 FBG levels of the three groups at week 10, prior to treatment.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/61357d00fe01abaa562ba1aa.jpeg"},{"id":98980231,"identity":"25922809-7bd4-4b21-a8b0-637b919fa732","added_by":"auto","created_at":"2025-12-25 06:39:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":549590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2A\u003c/strong\u003e FBG comparison between the D and the D + IL-18 groups on day 1 of treatment\u003c/p\u003e\n\u003cp\u003e2B FBG comparison on day 3 of treatment\u003c/p\u003e\n\u003cp\u003e2C FBG comparison on day 5 of treatment\u003c/p\u003e\n\u003cp\u003e2D FBG comparison on day 7of treatment\u003c/p\u003e\n\u003cp\u003e2E FBG comparison on day 9 of treatment\u003c/p\u003e\n\u003cp\u003e2F Body weight comparison between groups after day 9 of treatment\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/41f3dff63ecc8732c021921a.png"},{"id":98980230,"identity":"e15dc931-c10a-478e-8e09-c06689d406c5","added_by":"auto","created_at":"2025-12-25 06:39:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":465640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3A\u003c/strong\u003e Trend of FBG levels after treatment in each group\u003c/p\u003e\n\u003cp\u003e3B Blood glucose changes during fasting before the IPGTT in each group\u003c/p\u003e\n\u003cp\u003e3C IPGTT results for each group\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/8c0355a86c56ddcce47aa3fa.png"},{"id":99311662,"identity":"ca6df523-dec9-49cf-8cab-c8cbbb4ad0c2","added_by":"auto","created_at":"2025-12-31 16:16:24","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4ABC\u003c/strong\u003e HE staining in each group（the arrow points to the islet）\u003c/p\u003e\n\u003cp\u003e4DEF IHC staining in each group\u003c/p\u003e\n\u003cp\u003e4G Mean density of IHC in each group\u003c/p\u003e\n\u003cp\u003e4H Insulin-positive area of IHC in each group\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/517796cef96d6f99eaee2cb3.jpeg"},{"id":99311743,"identity":"2cc3e798-5897-4acd-9148-dd4a284bc31f","added_by":"auto","created_at":"2025-12-31 16:16:49","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":259679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5A-L\u003c/strong\u003e IF staining in each group;\u003c/p\u003e\n\u003cp\u003e5M Elevate caspase 3 mean density in IF staining for each group\u003c/p\u003e\n\u003cp\u003e5N Elevate caspase 3 area in IF staining for each group\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/eed1a294d3dcc484e45cac3d.jpeg"},{"id":98980235,"identity":"c332f609-fcb5-41d3-89e6-6c9e5030bed5","added_by":"auto","created_at":"2025-12-25 06:39:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":698512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e6A\u003c/strong\u003e Proportions of CD45+CD11b+ cells in the pancreas across each experimental group;6B Proportion of F4/80+ CD11b+ cells in the pancreas across each experimental group;6C Proportions of CD3-NK1.1+ in cells in the pancreas across each experimental group;6D Proportion of CD3+CD4+ cells in the pancreas across each experimet\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/6f98247282910a8f8ca5d0a9.png"},{"id":98980249,"identity":"4b47ecff-8aae-46c1-b745-79144a4fc8ef","added_by":"auto","created_at":"2025-12-25 06:39:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":225598,"visible":true,"origin":"","legend":"\u003cp\u003e7A Proportions of CD45+CD11b+ cells in the pancreas (n=3 per group);7BProportions of CD3-NK1.1- cells in the pancreas (n=3 per group);Samples were analyzed by flow cytometry Figure\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/d52944a3b98b980ea5a92fec.png"},{"id":99322913,"identity":"f121454c-d54f-4f86-8436-f2e6fa1a0397","added_by":"auto","created_at":"2025-12-31 16:44:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3223724,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8181438/v1/5f562302-325f-447c-9fc5-6bdb76642b60.pdf"}],"financialInterests":"","formattedTitle":"Effects of Exogenous Interleukin-18 on Islet Function and Immune Mechanisms in Type 1 Diabetic Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePediatric diabetes is an endocrine disorder that poses a significant threat to the health of children, with Type 1 Diabetes Mellitus (T1DM) being the predominant form [1]. In China, the prevalence of T1DM among children is estimated to range from 2 to 5 cases per 100,000 individuals, with an increasing trend toward earlier onset [2][3]. In 2022, the number of people living with T1D was estimated to be approximately 8.75\u0026nbsp;million worldwide, with 650,000 new cases that year. [4] The underlying cause of T1DM is damage of islets β-cells, causing severely impaired insulin secretion.\u003c/p\u003e \u003cp\u003eXian Z et al. [5] demonstrated through extensive animal studies that IL-18 plays a critical role in regulating insulin secretion and glucose metabolism. IL-18 acts through two key receptors: the IL-18 receptor (IL-18R) and the Na⁺/Cl⁻ cotransporter (NCC). Specifically, IL-18 is predominantly expressed in pancreatic α-cells, NCC in β-cells, and IL-18R in acinar cells. Deficiency in these receptors has been shown to reduce islet size, impair β-cell proliferation, and decrease insulin secretion. Moreover, under conditions such as diet-induced glucose intolerance or streptozotocin (STZ)-induced hyperglycemia, receptor deficiency exacerbates β-cell apoptosis. Also,the effect of interleukin-18 on pancreatic islet function is closely related to immune cells.Xian Z et al [5] considered acinar cell IL18r mediates IL18 activities inhibited macrophages expansion and inflammation in the hyperglycemic pancreas.These results indicate that endogenous IL-18 plays a critical protective role in maintaining islet homeostasis and immune mechanisms are involved.\u003c/p\u003e \u003cp\u003eConversely, Oikawa et al. [6] found that IL-18-expressing plasmid DNA increased the infiltration of CD4⁺ T cells and macrophages in the pancreas. Similarly, Li etal [7] identified immune cell infiltration, particularly CD4⁺ T cells and macrophages, as a central mechanism in T1DM pathogenesis in mouse models. These findings deduced exogenous IL-18 may could contribute to islet damage through pro-inflammatory pathways, while the effects and mechanisms of exogenous rIL18 on pancreatic function were not verified in live mice. To explore the differing effects of endogenous and exogenous IL-18, our study investigates the impact of exogenously administered IL-18 on islet function and immune responses in a mouse model of type 1 diabetes. We aim to elucidate the immune mechanisms underlying islet injury and dysfunction, with particular attention to the role of IL-18 in modulating immune cell infiltration. Additionally, we seek to clarify how different treatment course and cumulative dose of rIL-18 affect pancreatic islets, thereby providing a theoretical basis for developing IL-18-targeted therapeutic strategies for childhood diabetes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (18\u0026ndash;20 g, 5 weeks old) were obtained from Hangzhou Ziyuan Experimental Animal Technology Co., LTD. The mice were housed individually in a controlled environment, which was regulated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, with a 12-h light-dark cycle programmed from 08:00 to 20:00. Following one-week acclimatization period, 21 mice were randomly assigned into three groups: IL-18-treated diabetic group (D\u0026thinsp;+\u0026thinsp;IL-18; n\u0026thinsp;=\u0026thinsp;7);diabetic group (D; n\u0026thinsp;=\u0026thinsp;7), non-diabetic control group (CON; n\u0026thinsp;=\u0026thinsp;4).All animal studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of Anhui Provincial Hospital, affiliated with the First Affiliated Hospital of the University of Science and Technology of China.. (Approval No. 2025-N(A)-0115). All procedures were performed in strict compliance with the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003cp\u003eWe induced type 1 diabetes mellitus (T1DM) in 6-week-old male mice with a single intraperitoneal injection of freshly prepared STZ (180 mg/kg body weight) (Solarbio, China) dissolved in citrate buffer (pH 4.5), following overnight fasting. Blood glucose levels were measured after 4 weeks using a OneTouch II glucometer (Lifescan, Inc.) to confirm diabetic status (defined as fasting blood glucose [FBG]\u0026thinsp;\u0026gt;\u0026thinsp;16.6 mmol/L). After four weeks (when mice were 10 weeks old), diabetic mice were randomly divided into two subgroups: one received intraperitoneal injections of rmIL-18 (1 ug/mouse) (Medical \u0026amp; Biological Laboratories, Japan)every other day for 10 days, and the other received phosphate-buffered saline (PBS). Non-diabetic control mice received no treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Intraperitoneal glucose tolerance test (IPGTT)\u003c/h2\u003e \u003cp\u003eAfter a 12-hour fast(8AM-8PM), mice were administered glucose intraperitoneally (1 g/kg) ༈8PM༉. Blood glucose was then determined at intervals of 0 (baseline), 30, 60, and 120 minutes using Luo's glucose meter. Subsequently, the total area under the curve(AUC) for glucose was calculated according to the trapezoidal rule.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Organ harvesting\u003c/h2\u003e \u003cp\u003eThe pancreas were harvested after IPGTT, rinsed with PBS, and processed as follows: pancreatic portions were snap-frozen and stored at \u0026minus;\u0026thinsp;80℃ for further analysis; other portions were fixed in 10% formaldehyde for paraffin embedding and histological staining; remaining pancreatic tissue were processed into single-cell suspensions for flow cytometry analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Hematoxylin-eosin (HE) staining\u003c/h2\u003e \u003cp\u003eParaffin-embedded pancreatic tissue were sectioned at 5 \u0026micro;m, dewaxed with xylene, rehydrated in graded alcohols, and stained with 0.1% hematoxylin (G1120, Solarbio, Beijing, China) for 1 minute, followed by 0.5% eosin (G1120, Solarbio, Beijing, China) for 3 minutes, both at room temperature. Sections were mounted with glycerogelatin and imaged using an optical microscope (Axio Scope AI, Zeiss, Germany). Pancreatic morphology was observed and examined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunohischemistry (IHC) staining\u003c/h2\u003e \u003cp\u003eParaffin sections (5 \u0026micro;m) were cut into thick sections, which were processed through xylene dewaxing, graded alcohol rehydration, and antigen retrieval by heating in pH 6.0 citrate buffer (G1201, Servicebio, Wuhan, China) at 95℃ for 30 minutes. After cooling to room temperature, endogenous peroxidase activity was blocked using hydrogen peroxide. Sections were incubated overnight at 4\u0026deg;C with anti-insulin rabbit polyclonal antibody (pAB) (bsm-60010R, Bioss, Beijing, China), followed by a secondary antibody (G1302, Servicebio, Wuhan, China) for 60 minutes. After counterstaining with 0.1% hematoxylin (G1120, Solarbio, Beijing, China), slides were imaged using an optical microscope (Axio Scope AI, Zeiss, Germany). The proportion of insulin-positive cells within the islets were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003eParaffin sections (5 \u0026micro;m) were cut into thick sections, which were processed through xylene dewaxing, graded alcohol rehydration, and antigen retrieval by heating in pH 6.0 citrate buffer (G1201, Servicebio, Wuhan, China) to 95℃ and then immersed for 30 minutes and cooled to room temperature. Sections were incubated with anti-insulin mouse monoclonal antibody (mAb) (GB13121, Servicebio, Wuhan, China) and Anti-Cleaved-Caspase-3 rabbit pAb (GB11532, Servicebio, Wuhan, China) primary antibodies overnight at 4\u0026deg;C, then incubated with secondary antibodies (GB21303, GB22301, Servicebio, Wuhan, China) for 90 minutes. Cell nuclei were counterstained with DAPI (G1012, Servicebio, Wuhan, China), and slides were mounted with an anti-fluorescence quenching sealer (G1401, Servicebio, Wuhan, China). Fluorescence images were acquired with Zeiss Axio Imager M2microscope(Axio Imager M2,Zeiss,Germany).The proportion and intensity of insulin and cleaved caspase-3 expression were quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Flow cytometry (FC) analysis\u003c/h2\u003e \u003cp\u003eFresh spleen and pancreatic tissues were enzymatically digested using collagenase IV (BS165, Bioshap, Jiangsu, China) in RPMI-1640 medium (PM150110C, Wuhan, China) and passed through 40 \u0026micro;m filters. Cells suspensions were centrifuged at 600 g for 10 minutes, and red blood cells were lysed using RBC lysis buffer (R1010, Solarbio, Beijing, China). After washing with PBS, 100 \u0026micro;L of cell suspension was stained with the following antibodies: Fixable Viability Dye eFluor\u0026trade; 506 (65-0866-14, Thermo Fisher, USA), anti-CD45-Pacific blue (Proteintech), anti-CD3-PE (Proteintech), anti-CD4-APC (Proteintech), anti-NK1.1-FITC (Proteintech), anti-CD11b-Apc-cy7 (Proteintech), and anti-F4/80-PE-Texas Red (Proteintech). Samples were incubated on ice for 30 minutes in the dark, then analyzed using a BD Fortessa flow cytometer. Data were processed with FACSDIVA software (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eFBG levels and immune cell distributions were analyzed using one-way analysis of variance (ANOVA). Non-parametric data, including insulin and cleaved caspase-3 expression levels were analyzed using the Kruskal-Wallis test. All statistical analyses were performed with GraphPad Prism 8 software. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Exogenous rmIL-18 temporarily ameliorates fasting blood glucose (FBG) in T1DM mice, followed by deterioration after day 7.\u003c/p\u003e \u003cp\u003eExperimental T1DM models were successfully established:at day 0,we started modeling ,at day 28, FBG levels exceeded 16.7 mmol/L in both the D and D\u0026thinsp;+\u0026thinsp;IL-18 groups.At day 29, the mice of D\u0026thinsp;+\u0026thinsp;IL-18 groups received rmIL18(1ug/d)every other day for10 days,as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, and the experimental design is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB: the mice of D\u0026thinsp;+\u0026thinsp;IL-18 groups received rmIL18; D groups received PBS;Con groups receibed no treatment. FBG and body weight were monitored across all groups. Compared to the CON group, the body weights of the D and D\u0026thinsp;+\u0026thinsp;IL-18 groups were significantly decreased (Fig.\u0026nbsp;1C1), while FBG levels were significantly elevated (Fig.\u0026nbsp;1C2).\u003c/p\u003e \u003cp\u003eTreatment began at the start of week 10. On days 1, 3, and 5 of treatment, the FBG levels in the D\u0026thinsp;+\u0026thinsp;IL-18 group were significantly lower than those in the untreated diabetic group (D) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C. However, by day 7, no significant differences in FBG were observed between the two groups(p\u0026gt;0.05),as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. On day 9, a marked increase in FBG was observed in the D\u0026thinsp;+\u0026thinsp;IL-18 group compared with the D group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. By the end of treatment, No statistically significant difference in body weight as discernible between the D and D\u0026thinsp;+\u0026thinsp;IL-18 groups, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF.Overall, FBG levels in the D\u0026thinsp;+\u0026thinsp;IL-18 group initially decreased and then gradually increased, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA.in the D\u0026thinsp;+\u0026thinsp;IL-18 group,FBG levels showed an initial decline, followed by a gradual increase over time.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Exogenous rmIL-18 impairs islet function after day 7 of treatment\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBefore the IPGTT, mice were fasted for 12 hours. The decrease in FBG was more rapid in the D group than in the D\u0026thinsp;+\u0026thinsp;IL-18 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the IPGTT results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), the fasting (0 minutes) glucose level in the D\u0026thinsp;+\u0026thinsp;IL-18 group was significantly higher than in the D group. At all subsequent time points (15, 30, 60, 90, and 120 minutes), blood glucose remained higher in the D\u0026thinsp;+\u0026thinsp;IL-18 group. The AUC for glucose was significantly increased in the D\u0026thinsp;+\u0026thinsp;IL-18 group compared to both the D and CON groups (p \u0026lt; 0.05). Additionally, AUC was significantly lower in the CON group than in the D group (p \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To a certain extent, the findings revealed that the rIL-18 treated group exhibited a more pronounced impairment in islet function compared to the diabetic group.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 HE staining\u003c/h2\u003e \u003cp\u003eHistological analysis are shown in Figs.\u0026nbsp;4A\u0026ndash;C. In the CON group, islet morphology was well-preserved with clearly visible cell boundaries and minimal signs of denaturation or necrosis. In contrast, islets in both the D and D\u0026thinsp;+\u0026thinsp;IL-18 groups appeared irregular and structurally disrupted. The D\u0026thinsp;+\u0026thinsp;IL-18 group exhibited more pronounced cellular sparsity and vacuolation than the D group. A more severe disruption of the islet architecture was observed in diabetic mice following 7 days of rIL-18 treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 IHC staining\u003c/h2\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;4D\u0026ndash;F, islet size and β-cell proportion differed among groups. The control group exhibited the highest β-cell ratio and mean insulin density. These measures were reduced in the D group and further decreased in the D\u0026thinsp;+\u0026thinsp;IL-18 group, as quantified in Figs.\u0026nbsp;4G\u0026ndash;H. The already impaired insulin secretion in diabetic mice was further aggravated by a 7-day rIL-18 treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 IF staining\u003c/h2\u003e \u003cp\u003eTo evaluate apoptosis in pancreatic islets, co-staining for insulin (green), cleaved caspase-3 (red), and DAPI (blue) was performed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026ndash;L). The D\u0026thinsp;+\u0026thinsp;IL-18 group showed a higher proportion and intensity of cleaved caspase-3-positive cells compared to the D group. Both diabetic groups had significantly more caspase-3 expression than the CON group, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eM \u0026ndash;N. Apoptosis of pancreatic islets was markedly enhanced in diabetic mice following a 7-day course of interleukin 18.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Exogenous rmIL-18 promotes immune cell infiltration in the pancreas after day 7\u003c/h2\u003e \u003cp\u003eTo investigate the immune mechanism of islet dysfunction following rmIL-18 treatment, we analyzed immune cell infiltration in the pancreas. Flow cytometry assessed the proportions of T cells, NK cells, macrophages, and CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;cells. In the pancreas (Figs.\u0026nbsp;6A\u0026ndash;D), the proportions of CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;cells and NK1.1 cells were significantly elevated in the D\u0026thinsp;+\u0026thinsp;IL-18 group compared to both the D and CON groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B). No significant differences were found between the D and CON groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B). For CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells in the pancreas, the proportion was significantly lower in the D\u0026thinsp;+\u0026thinsp;IL-18 group compared to both the D and CON groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B). Thus, our findings demonstrate that rIL-18 drives the infiltration of macrophages and NK cells into the pancreatic tissue..\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIL-18 is a member of the IL-1 cytokine family that plays a centeral role in activating both innate and adaptive immunity [8]. It acts on Th1 cells, NK cells, B cells, and macrophages [9], and is implicated in the pathogenesis of multiple diseases [10]. Elevated IL-18 levels have been reported in conditions such as systemic lupus erythematosus [11], hypertension, chronic kidney disease [12], and type 2 diabetes and obesity [13]. However, research on the role of IL-18 in T1DM remains limited.\u003c/p\u003e \u003cp\u003eXian Z etal[5].demonstrated.endogenous IL-18 plays a critical protective role in maintaining islet homeostasis and macrophage were involved. no published studies have systematically investigated the effects of exogenous rmIL-18 on islet function and immune mechanisms in diabetic mice.Oikawa et al. [6] demonstrated that plasmid DNA expressing IL-18 accelerated the onset of diabetes in 4-week-old NOD mice, though the mechanism related to islet function and immune responses were not examined. Conversely, Rothe et al. [14] reported that exogenous IL-18 administration could prevent diabetes onset under certain conditions. Our findings showed that short-term rmIL-18 treatment led to a transient improvement in glycemic control during the first 7 days. This effect may be attributed to IL-18 acting on NCC in pancreatic islets, enhancing insulin gene expression, inhibiting macrophage infiltration, and reducing local inflammation, consistent with the findings of Zhang et al. [5].\u003c/p\u003e \u003cp\u003eOur study confirmed that rmIL-18 led to elevated FBG levels after 7 days of treatment, as evidenced by phenotypic analysis in diabetic mice. During a 12-hour fast before IPGTT, the rmIL-18-treated group declined significantly less than those in the untreated D group.Since the rate and extent of glucose reduction during fasting are closely related to pancreatic function[15], this observation supports the conclusion that islet function deteriorated following rmIL-18 administration.these findings was consistent with prior studies, such as by Frigerio et al. [16], who reported that IL-18 promotes islet cell death through pro-inflammatory cytokines and immune cell infiltration. Similarly, Dao et al. [17] demonstrated that IL-18 activates NK cells, which then interact with islet cells and induce programmed cell death.\u003c/p\u003e \u003cp\u003eThe pathogenesis of T1DM is primarily driven by the infiltration and attack of T cells and other immune cells on pancreatic β-cells, ultimately leading to their destruction and the development of an organ-specific autoimmune disease characterized by insulin deficiency. [18] Various immune cells, including T cells, B cells, NK cells, macrophages, and group 2 innate lymphoid cells (ILC2), have been shown to infiltrate pancreatic islets [19], where they interact with β-cells and promote their apoptosis. IL-18, a pro-inflammatory cytokine, plays key role in this process by acting on Th1 cells, NK cells, B cells, and macrophages [9], thereby modulating immune responses and contributing to target organ damage. In our study, flow cytometry revealed that IL-18 treatment after 7days significantly increased the proportion of pancreatic macrophages and NK cells, while T cells were markedly reduced, potentially due to the expansion of macrophages and NK cells. These immune changes, together with the observed phenotypic deterioration in islet structure and function following IL-18 treatment 7 days, support the conclusion that the decline in islet function is closely related to increased macrophage and NK cell infiltration and their cytotoxic interactions with pancreatic β-cells, which was consistent with the Li C[7] and Sophie L Walker[19].\u003c/p\u003e \u003cp\u003eIn summary, the effects of rmIL-18 on T1DM mice are complex and multifaceted. On one hand, IL-18 may enhance insulin synthesis by acting on the NCC receptor in pancreatic islets, thereby promoting insulin gene expression, inhibiting macrophage infiltration[4], in the early treatment courses and with smaller cumulative doses, rIL18 had similar effects to endogenous interleukin-18 in diabetic mice. On the other hand, as the treatment course and cumulative dose increased, rmIL-18 also facilitates the infiltration of immune cells, particularly macrophages and NK cells, into the pancreatic islets via IL-18R signaling, contributing to insulitis and accelerating islet apoptosis. The overall impact of exogenous IL-18 on islet function appears to depend on multiple factors, including treatment duration and cumulative dose, which together determine whether its net effect is protective or detrimental.\u003c/p\u003e \u003cp\u003eLimitation: Due to there was no precedent for exogenous interleukin 18 treatment of diabetic mice, we were unable to conduct IPGTT, collect tissue samples, or investigate the underlying mechanisms during the early phase of blood glucose reduction. As a result, the potential protective effects of exogenous IL-18 at the initial stage of treatment could not be fully explored. Further studies are needed to address this gap and further clarify the early immunometabolic responses to IL-18.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study investigated the effects of exogenous rmIL-18 on islet function in a mouse model of T1DM. During the first 7 days of treatment, FBG levels steadily declined. After 7 days of treatment, FBG levels began to rise again, and IPGTT revealed a deterioration in islet function. Histological analysis via HE and IHC staining showed more severe islet damage in the rmIL-18-treated group compared to the PBS-treated diabetic group, while IF confirmed increased islet cell apoptosis. Flow cytometry suggested that this deterioration may be associated with increased infiltration of macrophages and NK cells into the pancreas following rmIL-18 treatment.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental design was proposed by Xian Zhang and Shenggang Ding,all experiments were guided by Xian Zhang.Experiment completed,data acquisition, data analysis, and interpretation were carried out by Mei Xiong.The experimental data were processed by Li Ting ,all of them provided the final approval of the version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Anhui Provincial Hospital affiliated to the First Affiliated Hospital of University of Science and Technology of China and use Committee\u0026shy;【2025-N(A)-0115】.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the The National Natural Science Fund（52273113）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Anhui Provincial Hospital affiliated to the First Affiliated Hospital of University of Science and Technology of China and use Committee\u0026shy;【2025-N(A)-0115】.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eThe Subspecialty Group of Endocrinologic, Hereditary and Metabolic Diseases, the Society of Pediatrics(2020)Expert consensus on the standardized diagnosis and management of type 1 diabetes mellitus in Chinese children (2020)Chin J Pediatr 58, 6,447-454\u003c/li\u003e\n \u003cli\u003ePatterson CC(2019)Worldwide estimates of incidence, pevalence and mortality of type 1 diabetes in children and adolescents: Results from the International Diabetes Federation Diabetes Atlas, 9th edition.Diabetes research and clinical practice157,107842. DOI: 10.1016/j.diabres.2019.107842; PMID: 31518658\u003c/li\u003e\n \u003cli\u003eRabboneI, TraversiD, ScaioliG, etal (2020)Microbiota, epidemiological and nutritional factors related to ketoacidosis at the onset of type 1 diabete. ACTA DIABETOLOGICA57(11)1337-1349.DOI10.1007/s00592-020-01555-z\u003c/li\u003e\n \u003cli\u003eKossiva L, Korona A ,Kafassi N, etal (2022)Familial autoimmunity in pediatric patients with type 1 diabetes(T1D) and its associations with the severity of clinical presentation at diabetes diagnosis and with coexisting autoimmunity. HORMONES-INTERNATIONAL JOURNAL OF ENDOCRINOLOGY AND METABOLISM21(2)277-285.doi.org/10.1007/s42000-022-00358-x\u003c/li\u003e\n \u003cli\u003eZhang Xian,Luo Songyuan,Wang Minjie,etal(2022) IL18 signaling causes islet \u0026beta; cell development and delopment and insulin secretion via different receptors on acinar and\u0026beta; cells.Developmental Cell 57,1496-1511. DOI10.1016/j.devcel.2022.05.013\u003c/li\u003e\n \u003cli\u003eOikawa Y,ShimadaA, Kasuga A,etal(2003) Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice. J Immunol 171(11),5865-5875. DOI 10.4049/jimmunol.171.11.5865\u003c/li\u003e\n \u003cli\u003eLi C, Gao Q, Jiang H, etal(2022)Changes of macrophage and CD4(+) T cell in inflammatory response in type 1 diabetic mice. Sci Rep 12(1),14929. DOI10.1038/s41598-022-19031-9\u003c/li\u003e\n \u003cli\u003eSomm E Jornayvaz FR(2022) Interleukin-18 in metabolism:From mice physiology to human diseases FRONTIERS IN ENDOCRINOLOGY.2022,12:1664-2392. DOI10.3389/fendo.2022.971745\u003c/li\u003e\n \u003cli\u003eIhim SA,\u003csup\u003e\u0026nbsp;,\u003c/sup\u003eIhim\u003csup\u003e\u0026nbsp;\u003c/sup\u003eSD,Zian Z,etal(2022)Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment. Frontiers in Immunology 13, 1664-3224. DOI10.3389/fimmu.2022.919973\u003c/li\u003e\n \u003cli\u003eYasuda K, Nakanishi K, Tsutsui H(2019) Interleukin-18 in health and disease. Int J Mol Sci 20(3),649\u0026ndash;701. DOI10.3390/ijms20030649\u003c/li\u003e\n \u003cli\u003eRezaieyazdi Z, AkbariRad M, Saadati N, etal(2021) Serum interleukin-18 and its relationship with subclinical atherosclerosis in systemic lupus erythematosus. ARYA Atheroscler. 17(6),1\u0026ndash;6. DOI 10.22122/arya.v17i0.2126\u003c/li\u003e\n \u003cli\u003eThomas JM, Huuskes BM, Sobey CG, Drummond GR, Vinh A(2022) The IL-18/IL-18R1 signalling axis: Diagnostic and therapeutic potential in hypertension and chronic kidney disease. Pharmacol Ther 239:108191. DOI 10.1016/j.pharmthera.2022.108191\u003c/li\u003e\n \u003cli\u003eKaplanski Gilles(2018)Interleukin-18:Biological properties and role in disease, Pathogenesis Immunological Reviews 281,1, 0105-2896. DOI10.1111/imr.12616\u003c/li\u003e\n \u003cli\u003eRothe H, Hausmann A, Casteels K,etal(1999) IL-18 inhibits diabetes development in nonobese diabetic mice by counterregulation of Th1-dependent destructive insulitis. J Immunol 163,3,1230-1236\u003c/li\u003e\n \u003cli\u003eDeFronzo, R. A (2009) Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. \u003cem\u003eDiabetes\u0026nbsp;\u003c/em\u003e58(4), 773-795. DOL10.2337/db09-9028\u003c/li\u003e\n \u003cli\u003eFrigerio S, Holl\u0026auml;nder GA, Fau-Zumsteg U(2002)Functional IL-18 is produced by primary pancreatic mouse islets and NIT-1 beta cells and participates in the progression towards destructive insulitis. Horm Res 57(3\u0026ndash;4):94-104,0301-0163. DOI: 10.1159/000057959; PMID: 12006705\u003c/li\u003e\n \u003cli\u003eDao T, Mehal WZ, Crispe IN(1998) IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J Immunol 161(5),2217-2222. PMID: 9725214\u003c/li\u003e\n \u003cli\u003eZheng CA,Fu JF(2020) Pathogenesis of type 1 diabetes. Int J Pediatrics 47(4),274-278\u003c/li\u003e\n \u003cli\u003eWalker SL, Leete\u003csup\u003e\u0026nbsp;\u003c/sup\u003eP\u003csup\u003e\u0026nbsp;\u003c/sup\u003e, Boldison J(2025)Tissue Resident and Infiltrating Immune Cells: Their Influence on the Demise of Beta Cells in Type 1 Diabetes. Biomolecules 15,3,441?DOI: 10.3390/biom15030441;PMID: 40149976;PMCID: PMC11939886\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"exogenous, interleukin-18, islet function, immune mechanisms, T1DM","lastPublishedDoi":"10.21203/rs.3.rs-8181438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8181438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eType 1 diabetes mellitus (T1DM) is associated with the levels of\u003cstrong\u003e interleukin-18\u003c/strong\u003e（\u003cstrong\u003eIL-18\u003c/strong\u003e）. However, the effect of exogenous IL-18 and immune mechanisms involved have not yet been fully elucidated. This study aimed to inquire into\u003cstrong\u003e effects of exogenous IL-18\u003c/strong\u003e and \u003cstrong\u003eimmune mechanisms \u003c/strong\u003ein T1DM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: A \u003cstrong\u003etype 1 diabetes \u003c/strong\u003emouse model was established using \u003cstrong\u003ea single intraperitoneal injection \u003c/strong\u003eof freshly prepared STZ 180 mg per body weight. All the mice were all separated into three groups: diabetic mice treated with recombinant \u003cstrong\u003eIL-18 (1 ug/mouse) every other day\u003c/strong\u003e (D + IL-18 group)\u003cstrong\u003e;\u003c/strong\u003ediabetic mice treated with phosphate-buffered saline (D group); and a control group with non-diabetic mice receiving no treatment. Fasting blood glucose levels were measured throughout the treatment period. At the end of the 10 days treatment, an intraperitoneal glucose tolerance test was performed. To assess islet morphology and apoptosis, hematoxylin-eosin staining, immunohistochemistry, and immunofluorescence staining(insulin and elevate caspase 3 co-localization) were conducted. Flow cytometry was used to explore immune mechanisms of islet injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The type 1 diabetes mellitus model was successfully established. During the first 7 days of recombinant IL-18 treatment, the D + IL-18 group’s mice showed significantly lower fasting blood glucose levels than the D group’s mice (p \u0026lt; 0.05); however, glucose levels increased thereafter (p \u0026lt; 0.05). The intraperitoneal glucose tolerance test showed a larger area under the curve in the D + IL-18 group than in both the D and control groups (p \u0026lt; 0.05), indicating impaired glucose tolerance. Histological analysis revealed disrupted islet architecture and increased elevate caspase 3 expression, consistent with islet dysfunction. Flow cytometry demonstrated elevated proportions of CD11b⁺F4/80⁺ macrophages and CD3⁻NK1.1⁺ natural killer cells in the pancreas of IL-18–treated mice (p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Exogenous IL-18 temporarily ameliorated fasting blood glucose in T1DM mice, followed by deterioration in fasting blood glucose and islets function after day 7 ,potentially due to enhanced infiltration of CD11b⁺F4/80⁺ macrophages and CD3⁻NK1.1⁺ natural killer cells.\u003c/p\u003e","manuscriptTitle":"Effects of Exogenous Interleukin-18 on Islet Function and Immune Mechanisms in Type 1 Diabetic Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 06:39:47","doi":"10.21203/rs.3.rs-8181438/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-01-22T08:49:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-12-22T19:39:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-22T12:24:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Hormones","date":"2025-11-28T09:32:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T01:54:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hormones","date":"2025-11-25T06:10:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d041889c-07e4-4cc2-a2bb-16ba2620cca9","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T11:30:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-25 06:39:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8181438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8181438","identity":"rs-8181438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}