T helper 17 cells and interleukin-17 immunity in type 1 diabetes: From pathophysiology to targeted immunotherapies

Abstract

Type 1 diabetes (T1D) is a chronic organ-specific autoimmune disorder characterized by a progressive loss of the insulin-secreting pancreatic beta cells, which ultimately results in insulinopenia, hyperglycemia and lifelong need for exogenous insulin therapy. In the pathophysiological landscape of T1D, T helper 17 cells (Th17 cells) and their hallmark cytokine, interleukin (IL)-17, play pivotal roles from disease onset to disease progression. In this narrative mini-review, we discuss the dynamic interplay between Th17 cells and IL-17 in the context of T1D, providing insights into the underlying immunologic mechanisms contributing to the IL-17-immunity-mediated pancreatic beta-cell destruction. Furthermore, we summarized the main animal and clinical studies that investigated Th17- and IL-17-targeted interventions as promising immunotherapies able to alter the natural history of T1D.

Key Words: Type 1 diabetes; T helper 17 cells; Interleukin-17; T helper 1 cells; Regulatory T cells; Anti-interleukin-17 treatment; Th17-targeted treatment

Core Tip: Recent research has identified Th17 cells and their primary effector cytokine, interleukin (IL)-17, as key players in the pathophysiology of type 1 diabetes (T1D). T helper 17 cells (Th17 cells) contribute to the autoimmune responses directed against pancreatic beta cells in T1D, promoting insulitis and beta-cell destruction. Understanding the role of Th17 cells and IL-17 in T1D pathophysiology offers new opportunities for targeted immunotherapies, which aim to modulate the altered immune responses and preserve residual beta-cell function in presymptomatic and new-onset T1D. Therefore, Th17 cell- and IL-17-targeted interventions may potentially represent future disease-modifying therapies able to improve clinical outcomes in patients with T1D at different stages of the disease.

INTRODUCTION

Type 1 diabetes (T1D) is a chronic organ-specific autoimmune disorder characterized by a gradual loss of insulin-secreting pancreatic beta-cells, which ultimately results in insulinopenia, hyperglycemia and lifelong need for exogenous insulin therapy[1]. Moreover, T1D is a polygenic disease influenced by various environmental factors. Such environmental factors include viral infections (i.e., infections caused by enteroviruses), as well as hypovitaminosis D, patterns of infant feeding, omega-3 polyunsaturated fatty acid deficiency, and many others[2-5].

The risk of T1D is increased by specific human leukocyte antigen (HLA) DR/DQ alleles, with the DR3/4-DQ8 heterozygous genotype being associated with the highest risk for T1D [6]. Globally, the incidence of T1D is on the rise, particularly in the pediatric population[7]. Given that genetic factors alone cannot account for such a rapid increase in the incidence of T1D, environmental factors must be implicated in this epidemiological trend[8]. In particular, T1D incidence has been reported to increase by approximately 3.4% per year during the last three decades[9,10]. The highest incidence rates of T1D have been observed in Scandinavia, with Finland and Sweden showing an age-standardized incidence of T1D above 44 per 100000 per year in children aged 0-14 years[11]. This alarming epidemiological trend underscores the need for a deeper understanding of the pathophysiology of T1D, as well as the need for the development of effective prevention and treatment strategies for this disease.

T1D has a profound socioeconomic and psychological impact[12,13], negatively affecting the patients’ quality of life and also posing a significant burden on the healthcare systems[14,15]. Proper management of T1D requires continuous glucose monitoring, exogenous insulin administration and appropriate dietary planning, which can be challenging and stressful for patients and their families[14,16]. The chronic nature of the disease and the risk of chronic complications (such as retinopathy, nephropathy, neuropathy and cardiovascular disease) further exacerbate these challenges. Moreover, the clinical onset of T1D often occurs during childhood or adolescence, thus negatively impacting educational and social experiences[17].

At the present time, a definitive biological cure for T1D is still not available. However, advancements in comprehending the immunologic mechanisms underlying T1D pathophysiology have paved the way for developing potential targeted immunotherapies. A promising therapeutic approach focuses on the role of T helper 17 cells (Th17 cells) and their primary cytokine [interleukin (IL)-17]. Even though T1D has been classically considered a type 1 T helper (Th1) cell-mediated disease, a growing body of evidence supports the crucial role of Th17 cells and IL-17 immunity in the pathophysiology of this disease[18,19].

Th17 cells are highly pro-inflammatory effector T cells that produce elevated amounts of IL-17A, IL-17F, IL-21, and IL-22[20]. Th17 cells have been associated with the pathophysiology of different autoimmune diseases[20]. In this regard, Th17 cells have been identified as critical players in the autoimmune response that leads to beta-cell destruction in T1D[21]. Experimental studies in non-obese diabetic (NOD) mice have shown that interventions targeting Th17 cells and IL-17 can modulate the progression of T1D, suggesting that inhibition of Th17 cells and inhibition of IL-17 production could be viable therapeutic strategies for T1D[21]. Additionally, manipulating regulatory T cells (Tregs), which are critical for maintaining immune tolerance, offers another therapeutic avenue in this direction. Indeed, enhancing the function of Tregs and/or expanding the Treg population could help suppress the T1D-related autoimmune attack directed against pancreatic beta cells[22].

Despite the advancements mentioned above, several therapeutic challenges remain unresolved. One major issue is the heterogeneous nature of T1D, with a high interindividual variability in disease progression and response to immunotherapies observed among T1D patients[23-25]. This heterogeneity complicates the development of disease-modifying therapies that would be effective in all patients affected by T1D. Moreover, while IL-17 inhibitors and Treg-based therapies showed promise in preclinical studies[21,22], their long-term safety and efficacy in humans need to be evaluated in clinical trials. Another significant therapeutic challenge is the need for early intervention in T1D. Indeed, a substantial proportion of beta cells is already lost by the time T1D is clinically diagnosed, thus limiting the potential efficacy of delayed therapeutic interventions in reversing the disease progression[25]. Therefore, early diagnosis of T1D and prompt post-diagnosis interventions are critical for achieving successful therapeutic outcomes related to targeted immunotherapies[24].

In the present narrative mini-review, we discuss the role of Th17 cells and their primary cytokine IL-17 as critical drivers of beta-cell autoimmunity in T1D. Indeed, targeting the IL-17 signaling pathway may alter the clinical course of T1D and protect pancreatic beta cells from immune-mediated destruction. In particular, this mini-review aims to elucidate the mechanisms by which Th17 cells and the pro-inflammatory cytokine IL-17 contribute to T1D pathophysiology and to evaluate the therapeutic potential of IL-17 inhibitors in both preclinical and clinical settings. Importantly, a better understanding of these mechanisms may help identify novel therapeutic targets that could be used to develop more effective and tailored immunotherapies for T1D.

SEARCH STRATEGY

To explore the role of Th17 cells and IL-17 in T1D, we conducted a literature search across four major scientific databases: PubMed, MEDLINE, Scopus and Web of Science. The literature search regarded articles published from January 1990 to July 2024, ensuring the inclusion of preclinical and clinical research articles. Keywords used included the following: "Th17 cells", "IL-17", "Type 1 Diabetes", "pathophysiology", and "immunotherapy". We used the following Boolean operators: ("Th17 cells" OR "T-helper 17 cells") AND ("IL-17" OR "Interleukin-17") AND ("Type 1 Diabetes" OR "T1D" OR "Type I Diabetes") AND ("pathophysiology" OR "mechanism" OR "immune response") AND ("immunotherapy" OR "treatment" OR "therapy").

The initial search results were screened for relevance using specific inclusion and exclusion criteria. Inclusion criteria were the following: (1) Original research articles, clinical trials, and systematic or narrative review articles; (2) Studies focusing on the role of Th17 cells and IL-17 in the pathophysiology, immune mechanisms or therapeutic outcomes of T1D; and (3) Articles available in English. Exclusion criteria were the following: (1) Studies not directly related to T1D, Th17 cells or IL-17; (2) Conference abstracts, conference proceedings, and articles lacking sufficient data on Th17 or IL-17 in T1D; and (3) Duplicate articles identified across multiple databases.

The initial search yielded 522 articles (Figure 1). After article titles and abstracts were screened for relevance, we included original research articles, clinical trials, and review articles that mainly focused on the involvement of Th17 cells and IL-17 in T1D pathophysiology and treatment. The final version of the present manuscript included 99 key articles, thus providing a solid foundation for this narrative review.

Figure 1 Results of the literature search employed for the identification of studies on the role of T helper 17 cells and interleukin-17 in type 1 diabetes.

IMMUNOPATHOLOGY OF T1D

The pathogenesis of T1D involves a complex network of relationships between pancreatic beta cells and the innate and adaptive immune systems.

Immune cells involved in T1D pathophysiology

With regard to T1D pathophysiology, it is thought that antigen-presenting cells bearing surface beta-cell antigens migrate to local pancreatic lymph nodes, where they present those antigens to autoreactive CD4+ T lymphocytes. CD4+ T lymphocytes then mediate the recruitment of autoreactive cytotoxic CD8+ T lymphocytes that migrate to the pancreatic islets and recognize major histocompatibility complex class I-restricted islet autoantigens on the surface of beta cells[25], while Tregs exhibit defects in their ability to suppress the proliferation of autoreactive T cells[26]. CD8+ T lymphocytes exert cytotoxic effects through different effector mediators, particularly cytokines released by Th1 cells such as interferon (IFN)-γ[27,28].

The pathologic process of pancreatic islet infiltration by immune cells is also referred to as "insulitis" and represents the histopathological hallmark of the autoimmune destruction of pancreatic beta cells in T1D[29]. Even though autoreactive CD8+ cytotoxic T cells represent the most abundant immune cell infiltrating the pancreatic islets of T1D subjects, CD4+ T lymphocytes, B lymphocytes and macrophages are also present, particularly in young children[27,30].

B lymphocytes, Th17 cells and follicular helper T cells also play an important role in T1D pathophysiology[18,31]. In particular, B lymphocytes represent the source of islet autoantibodies in T1D, which serve as diagnostic biomarkers for the disease[32,33]. Another component of the immune dysregulation in T1D is represented by the defective function of Tregs, which fail to suppress beta-cell autoimmunity[34]. In fact, various studies demonstrated that patients with T1D exhibit defects in the ability of Tregs to suppress the activity and proliferation of autoreactive CD4+ and CD8+ T cells[26,35,36].

Beta-cell abnormalities have also been proposed as possible drivers of T1D-related beta-cell autoimmunity. It has been found that HLA class I antigens and β2-microglobulin (a second component essential for the generation of functional HLA class I complexes) are overexpressed in the insulin-containing islets of patients with T1D, potentially promoting early disease progression through the effective engagement of cytotoxic CD8+ T lymphocytes specific to defined islet antigens[37]. Beta-cell autoimmunity, as evidenced by the appearance of circulating islet autoantibodies, is usually present months to years before the clinical onset of T1D[38].

Beta-cell autoimmunity and islet autoantibodies in T1D

Islet autoantibodies include glutamic acid decarboxylase autoantibodies (GADA), tyrosine phosphatase-related islet antigen 2 autoantibodies (IA-2A), zinc transporter 8 autoantibodies (ZnT8A), insulin autoantibodies (IAA), and pancreatic islet cell antibodies (ICA)[39,40]. Moreover, tetraspanin-7 has recently been identified as a novel target of autoimmunity in T1D, allowing its possible exploitation for T1D prediction and immunotherapy[41].

Stages of T1D

Genetic susceptibility represents the starting point in the natural history of T1D, which then, upon exposure to one or more environmental triggers, follows a specific evolution characterized by three distinct sequential stages, namely:

Stage 1 (islet autoimmunity): Presence of islet autoantibodies, normoglycemia, and absence of clinical symptoms.

Stage 2 (dysglycemia): Presence of islet autoantibodies, dysglycemia [as evidenced by impaired fasting glucose, abnormal oral glucose tolerance test (OGTT), and/or glycated hemoglobin (HbA1c) values ≥ 5.7%], and absence of clinical symptoms.

Stage 3 (symptomatic disease): Presence of islet autoantibodies, hyperglycemia, and presence of clinical symptoms of T1D, such as polyuria, polydipsia, fatigue, weight loss, and/or diabetic ketoacidosis[42,43]. Figure 2 illustrates these stages (Figure 2A) along with the concomitant autoimmune processes accounting for beta-cell dysfunction and loss (Figure 2B).

Figure 2 Natural history of type 1 diabetes and role of T helper 17 cells/interleukin-17 immunity in the pathophysiology of type 1 diabetes. Type 1 diabetes (T1D) stages are characterized by a progressive decline in beta-cell mass and function due to underlying autoimmune processes. A: Depicts the progressive decline in functional beta-cell mass throughout the sequential stages of T1D (stages of islet autoimmunity, dysglycemia and symptomatic disease); B: Illustrates the immune dysregulation observed in T1D, emphasizing the imbalance between pro-inflammatory T helper 17 (Th17) cells and regulatory T cells (Tregs). Th17 cells (involved in interleukin-17 production) promote beta-cell destruction, while Tregs (which physiologically maintain immune tolerance) are poorly represented and/or functionally impaired. This imbalance contributes to the immune-mediated destruction of insulin-producing beta cells and may be targeted by immunotherapies employed for the treatment of T1D. CD: Cluster of differentiation; T1D: Type 1 diabetes; IL-17: Interleukin-17; Th-17: T helper 17; FOXP3: Forkhead box P3; RORγt: Retinoic acid-related orphan receptor gamma t; Tregs: Regulatory T cells. Figure 2 was partly created with images adapted from Servier Medical Art licensed under the Creative Commons Attribution 4.0 International License (CC BY 4.0) (https://smart.servier.com/).

With regard to T1D pathophysiology, significant interindividual heterogeneity also exists regarding the extent of residual beta-cell function. Indeed, some patients with long-standing T1D maintain detectable levels of serum C-peptide (a surrogate marker of endogenous insulin secretion) and show the persistence of insulin-containing pancreatic islets even after many years from the disease diagnosis[29,44-49]. These findings are highly relevant since retention of endogenous insulin secretion has been associated with better glucose control, lower glucose variability, along with lower risk of hypoglycemia and chronic complications of diabetes such as retinopathy and nephropathy[50-53].

TH17 CELLS AND REGULATORY T CELLS IN T1D

Th17 cells in T1D

Over the last decade, studies have hypothesized that Th17 CD4+ cells might play a pathogenic role in various chronic inflammatory and autoimmune conditions, including T1D and inflammatory bowel disease[21,54]. Given that IL-17 is the main effector molecule secreted by Th17 cells, an experimental study investigated whether anti-IL-17 antibodies or recombinant IL-25, which directly suppresses Th17 responses[55], could alter the natural history of diabetes in NOD mice[56], a well-established animal model of T1D[57]. Although the interventions mentioned above did not prevent autoimmune diabetes when started at an early stage, they rapidly halted disease progression at the stage of insulitis by reducing peri-islet T-cell infiltrates and GAD65 antibody production and by increasing the frequency of Tregs in NOD mice[56]. In addition, IL-25 therapy was superior to anti-IL-17 during mature diabetes since it determined a period of remission from new-onset diabetes in 90% of the treated animals[56]. These findings suggest that IL-17 neutralization can prevent the onset of autoimmune diabetes after the development of insulitis but not earlier, indicating an interference with the effector phase of T1D. Interestingly, another experimental study examined the effects of T cell receptor transgenic T cells directed against GAD sequence 206-220 (designated GAD2), representing a late-stage epitope[58]. Those transgenic cells produced IFN-γ, a cytokine that inhibits Th17 cells. The study showed that the induction of splenic IFN-γ mediated the clearance of pancreatic cell infiltration, the promotion of beta-cell division and the restoration of normoglycemia in NOD mice at the prediabetic stage by inhibiting IL-17 production[58]. Accordingly, IFN-γ neutralization caused a significant increase in Th17 cell frequency and rendered the treatment nonprotective[58]. Researchers suggested that effective presentation of GAD2 peptide under non-inflammatory conditions may protect against T1D at advanced stages and that IFN-γ induced by an adjuvant-free antigen, in contrast with its typical inflammatory function, can restore normoglycemia by localized bystander suppression of pathogenic IL-17-producing cells[58].

Another study identified a tumor necrosis factor alpha (TNF-α)-dependent mechanism through which Th17 cells can contribute to the pathophysiology of autoimmune diabetes in NOD mice[59]. The authors showed that islet-specific Th17 cells were diabetogenic independently of IL-17 and exhibited an inflammation-induced Th17-to-Th1 reprogramming elicited by Th1 cells. Nevertheless, while an inability to generate Th1 cells due to Stat4, Ifngr, and Ifng deficiencies did not prevent diabetes, TNF-α mediated diabetes in response to either Th17 cells or Th1 cells[59].

Moreover, IL-17A has been shown to augment IL-1β+IFN-γ- and TNF-α+IFN-γ-induced chemokine mRNA and protein expression, as well as apoptosis in human pancreatic islets, suggesting that it potentially contributes to T1D pathophysiology through exacerbation of beta-cell apoptosis and enhanced local production of chemokines[60].

Further studies have also highlighted the role of IL-17 immunity in T1D pathophysiology, thus pointing to a potential therapeutic strategy targeting the IL-17 pathway for the treatment of T1D. Upregulation of Th17 immunity [characterized by increased IL-17 secretion, enhanced expression of IL-17, IL-22, retinoic acid-related orphan receptor C isoform 2 (RORC2) and forkhead box P3 (FOXP3)] has also been found in peripheral blood T cells from children with newly diagnosed and long-standing T1D[19]. Moreover, circulating memory CD4+ cells from T1D children have been shown to exhibit the same pattern of IL-17, IL-22 and FOXP3 mRNA upregulation, thus supporting the in vivo activation of IL-17 signaling pathway[19].

Similarly, it has been reported that children with new-onset T1D exhibit an increased proportion of both IL-17-secreting CD4+ and CD8+ T cells, as well as an increased proportion of CD45RA(-) CD25(int) FOXP3(low) cells that are not suppressive and produce significantly more IL-17 than other FOXP3+ Tregs[61].

Another study investigating the peripheral blood CD4+ T-cell responses to beta-cell autoantigens (GAD65, insulinoma-associated protein, and proinsulin peptides) in patients with new-onset T1D documented that 54% of subjects exhibited IL-17 reactivity to one or more beta-cell peptides[62]. Authors also demonstrated that IL-17 drives a significant enhancement of IL-1β/IFN-γ- and TNF-α/IFN-γ-induced apoptosis in human islets, rat beta-cells and INS-1E cells, in combination with a substantial upregulation of beta-cell IL-17 receptor A expression through the activation of the transcription factors STAT1 (Signal transducer and activator of transcription 1) and nuclear factor (NF)-κB[62]. These findings highlighted the existence of a specific pathway to beta-cell death involving IL-17, STAT1 and NF-κB, confirming the potential role of IL-17 as a novel T1D biomarker and promising therapeutic target. Importantly, a pro-inflammatory cytokine milieu promoting Th17 differentiation may be responsible for the upregulation of IL-17 immunity and the elevated circulating IL-17 levels found in patients with T1D[63-65].

The balance between Th17 cells and Tregs in T1D

The important role of the balance between Th17 cells and Tregs in T1D pathophysiology cannot be overlooked. In fact, a normal balance between Th17 cells and Tregs (which have opposing functions) is essential for the maintenance of immune homeostasis and for the prevention of autoimmunity[66,67]. In this regard, T1D-related upregulation of IL-17 immunity might result from Treg cell dysfunction[64,68,69](Figure 2B). Moreover, an imbalance between Th17 cells and Tregs has also been linked to chronic inflammatory and autoimmune diseases other than T1D[66,67,70].

Studies have explored the possible mechanisms by which Tregs control Th17 cell responses. According to a study conducted by Chaudhry et al[71], Foxp3 binding to STAT3, a critical transcription factor for Th17 differentiation, allows CD4+ Tregs to regulate the Th17 immune response in mice. Given that Foxp3 overexpression has been shown to inhibit Retinoic acid-related orphan receptor gamma t (RORγt)-mediated IL-17A mRNA transcription via direct interaction with RORγt, it has been proposed that Foxp3 induction represents the mechanism accounting for the suppression of Th17 cells[72]. These findings confirm the hypothesis that low Foxp3 expression could result in ineffective modulation of Th17 differentiation[73]. In addition, IL-17 immunity may also play a role in Treg dysfunction, since anti-IL-17 treatment significantly increases Treg frequency in NOD mice[56].

In their systematic review, Brenu et al[74] examined circulating biomarkers during T1D progression , describing the transcription factors RORC, FOXP3, T-bet and GATA3, as well as mRNA levels of several genes (IL-17A, IL-17F, IFN-γ, IL-9, Foxp3) as potential biomarkers for T1D diagnosis and progression. Interestingly, Bi et al[75] showed that omega-3 polyunsaturated fatty acids sharply reduced the incidence of diabetes mellitus in NOD mice partly by decreasing the proportion of Th17 cells and increasing the proportion of Tregs.

In summary, Th17 cells play an important role in T1D pathophysiology. These cells contribute to autoimmune responses directed against pancreatic beta cells by promoting immune-mediated beta-cell destruction. Therefore, understanding the mechanisms by which Th17 cells influence T1D development and progression, including the interactions of Th17 cells with various cytokines and other immune cells, can provide valuable insights into the identification of novel therapeutic targets to halt beta-cell autoimmunity in T1D.

ANIMAL STUDIES ON TH17- AND IL-17-TARGETED THERAPIES FOR T1D

Experimental studies in animal models of T1D have provided remarkable insights into the therapeutic potential of interventions targeting the Th17 pathway. Animal models of T1D, particularly NOD mice, have been instrumental in testing various strategies to inhibit Th17 cell differentiation, as well as monoclonal antibodies directed against IL-17 or IL-17 receptors. Studies have demonstrated that targeting the Th17 pathway can reduce insulitis, preserve beta-cell function and regulate blood glucose levels in animal models of T1D, highlighting the potential of Th17- and IL-17-targeted therapies in modifying the natural history of this disease[21,76].

These findings provide the basis for further preclinical and clinical investigations on Th17- and IL-17-targeted therapies in T1D. In this regard, evaluating the therapeutic efficacy of newly identified ROR-specific synthetic ligands that act on RORα and RORγt is also highly interesting, as RORα and RORγt play critical roles in Th17 cell development[77]. For example, Solt et al[78] treated NOD mice with the selective RORα/γ inverse agonist SR1001. The treated mice showed a substantial reduction in the incidence of diabetes and insulitis. Moreover, SR1001 raised the frequency of CD4+ Foxp3+ Tregs, decreased the production of islet autoantibodies, and reduced the expression of pro-inflammatory cytokines, especially the expression of Th17-mediated cytokines[78].

However, studies conducted in diabetic mice models present challenges regarding the prediction of the therapeutic efficacy of IL-17 inhibitors. The predominant effector T cells in NOD mice seem to be Th1 cells that produce IFNγ. There is also a debate over Th17 cell function in the NOD mice model. Although some studies indicated that IL-17 and IL-21 play a role in the development of autoimmune diabetes in NOD mice[56,79], other studies suggested that Th17 cells play an indirect role in T1D development, presumably by conversion to Th1-like cells[80,81]. A study published by Joseph et al[82] suggested that IL-17 may play a dispensable role in the pathophysiology of autoimmune diabetes. By using lentiviral transgenesis to generate NOD mice in which IL-17 is silenced via RNA interference, the authors demonstrated that loss of IL-17 did not affect the frequency of spontaneous or cyclophosphamide-induced diabetes. On the contrary, IL-17 silencing in transgenic NOD mice was sufficient to decrease the severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. This is consistent with reports that IL-17 deficiency exerts a protective role in this experimental model of multiple sclerosis[82].

Table 1 summarizes the main studies using Th17- and IL-17-targeted therapies in animal models of autoimmune diabetes and related chronic complications[56,78,80,83-87].

Table 1 Summary of the main studies on the use of Th17- and interleukin-17-targeted therapies in animal models of autoimmune diabetes and related chronic complications.

Animals	Induced experimental diabetes and/or related chronic complication	Intervention	Results	Ref.
NOD/LtJ and NOD-RAG−/− mice (NOD.129S7-Rag1tm1Mom/J)	NOD mice as a model of spontaneous autoimmune diabetes	Inhibition of Th17 cells using both neutralizing anti-IL-17 antibodies and recombinant IL-25	Reduction in peri-islet T-cell infiltrates and GAD65 antibody production; increase in the frequency of regulatory T cells. IL-25 therapy was superior to anti-IL-17 treatment mature diabetes since it determined a period of remission from new-onset diabetes in 90% of the treated animals	Emamaullee et al[56], 2009
C57BL/6 mice	STZ-induced diabetes; signs of diabetic retinopathy	Anti-IL-17A intravitreal injection treatment. Mouse IL-17A antibody (a neutralizing, monoclonal IgG1 antibody binding to IL-17A, and inhibiting IL-17A activity) was injected intravitreally	Decrease in retinal inflammation and ZO-1 degradation within the retinal endothelium	Zhou et al[83], 2023
Male C57BL/6J inbred mice	Autoimmune diabetes induced by multiple low doses of STZ	TCV aimed to inhibit autoimmune diabetes in mice through the suppression of Th17 cells	TCV decreased hyperglycemia, preserved the number of healthy pancreatic islets and increased insulin production in the pancreatic islets; moreover, TCV led to reduced production of both IL-17 and IL-23 in intrapancreatic infiltrating lymphocytes via marked inhibition of mRNA level of RORγt and Stat3 phosphorylation	Wang et al[84], 2011
NOD/SCID mice	Mouse model of T1D	Authors investigated whether mouse Th17 cells with specificity for an islet antigen can induce diabetes upon transfer into NOD/SCID recipient mice. Authors also investigated whether use of neutralizing IL-17-specific antibodies can prevent autoimmune diabetes	Induction of diabetes in NOD/SCID mice through adoptive transfer of Th1 cells from BDC2.5 transgenic mice was prevented by treatment of the recipient mice with a neutralizing IFN-γ-specific antibody, suggesting a major role of Th1 cells in the induction of disease in this animal model of T1D. Transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients with similar rates of onset as it was observed after transfer of Th1 cells. However, treatment with neutralizing IL-17-specific antibodies did not prevent the disease. Instead, the transferred Th17 cells, which were completely devoid of IFN-γ at the time of transfer, rapidly converted to secrete IFN-γ in the NOD/SCID recipient mice. These results indicate the existence of a plasticity of Th17 cell commitment towards a Th1-like profile	Bending et al[80], 2009
Female NOD/LtJ mice	Mouse model of T1D	Administration of the selective RORα/γ inverse agonist SR1001	SR1001-treated mice showed a substantial reduction in the incidence of diabetes and insulitis. SR1001 raised the frequency of CD4+ Foxp3+ Tregs, decreased the production of islet autoantibodies, and reduced the expression of pro-inflammatory cytokines (particularly the expression of Th17-mediated cytokines)	Solt et al[78], 2015
Male 8-week-old C57BL/6 mice	Intraperitoneal injections of STZ to induce diabetic retinopathy	Diabetic retinopathy model mice were treated with anti-IL-17A or anti-IL-17RA monoclonal antibodies administered into the vitreous cavity	Intravitreal injections of anti-IL-17A or anti-IL-17RA monoclonal antibodies reduced Müller cell dysfunction, vascular leakage, vascular leukostasis, tight junction protein downregulation, as well as ganglion cell apoptosis within the retina	Qiu et al[85], 2017
Ins2Akita (Akita) mice	T1D model with a spontaneous mutation in the insulin 2 gene leading to beta-cell apoptosis. STZ was also used to induce a diabetic model in MIN6 cells, a mouse insulinoma cell line	Anti-IL-17RA-neutralizing antibody used in MIN6 cells	IL-17A-knockout Akita mice showed reduced blood glucose concentrations and raised serum insulin levels. IL-17A deficiency decreased the production of the pro-inflammatory cytokines TNF-α, IL-1β, and IFN-γ in Akita mice, whereas IL-17RA expression in MIN6 cells was upregulated by IL-17A. IL-17A enhanced the expression of TNF-α, IL-1β, IFN-γ and iNOS, while it further augmented the STZ-induced inflammatory responses in MIN6 cells. IL-17A exacerbated STZ-induced MIN6 cell apoptosis and impairment in insulin secretion. Blocking IL-17RA with the use of an anti-IL-17RA-neutralizing antibody reduced all the aforementioned deleterious effects exerted by IL-17A on MIN6 cells. Overall, IL-17A deficiency alleviated hypoinsulinemia, hyperglycemia and inflammatory response in Akita mice. IL-17A exerted deleterious effects (alone and in combination with STZ) on pancreatic beta cells through the IL-17RA pathway	Qiu et al[86], 2021
Male WT C57BL/6 mice	Intraperitoneal administration of STZ to induce diabetes and diabetic nephropathy	Use of IL-17 knockout mice or administration of neutralizing anti-IL-17 monoclonal antibody	IL-17-deficient mice were protected against the progression of diabetic nephropathy, showing reductions in glomerular damage, albuminuria, macrophage accumulation, and renal fibrosis at 12 and 24 wk. Administration of the anti-IL-17 monoclonal antibody to diabetic wild-type mice exerted similar protective effects. IL-17 deficiency also mitigated the up-regulation of pro-inflammatory and pro-fibrotic genes such as TNF-α, IL-6, CCL2, TGF-β and CXCL10 in diabetic kidneys. In vitro co-stimulation with recombinant IL-17 and high glucose exerted synergistic effects in increasing the expression of pro-inflammatory genes in cultured renal tubular cells and podocytes	Ma et al[87], 2019

NOD mice: Non-obese diabetic mice; T1D: Type 1 diabetes; IL: Interleukin; Th-17: T helper 17; FOXP3: Forkhead box P3; RORγt: Retinoic acid-related orphan receptor gamma t; Tregs: Regulatory T cells; STZ: Streptozotocin; TCV: T-cell vaccination; Stat3: Signal transducer and activator of transcription 3; ZO-1: Zonula Occludens-1; IFN: Interferon; TNF: Tumor necrosis factor; TGF: Transforming growth factor; iNOS: Inducible nitric oxide synthase; SCID: Severe combined immunodeficiency; WT: Wild-type.

CLINICAL STUDIES ON TH17- AND IL-17-TARGETED THERAPIES FOR T1D

Since the description of the IL-23/IL-17 immune pathway[88], studies have been conducted to bring innovative therapeutic approaches into clinical practice. These findings are promising for the treatment of chronic inflammatory and autoimmune conditions such as rheumatoid arthritis, inflammatory bowel disease and T1D[21,76,89-91].

As mentioned above, preclinical research has shown that autoimmune diabetes in NOD mice can be controlled by directly suppressing Th17 cells or neutralizing IL-17 activity. Although clinical studies on anti-Th17 and anti-IL-17 treatments in T1D are still in the early stages, they have begun to provide essential data on the safety and potential benefits of such therapeutic approaches. While it was originally thought that IL-23 controls pathogenic IL-17 synthesis and that these cytokines represent "duplicate" targets, subsequent clinical results have suggested that this is not the case[92]. The success of targeting the IL-17 pathway using p40 blockade in psoriasis made this therapeutic approach potentially attractive even for T1D[93].

Initial clinical trials have focused on assessing the safety profile and pharmacokinetics of anti-Th17 therapies, as well as their ability to preserve circulating C-peptide levels in T1D patients. While research data are preliminary, they suggest that anti-Th17 therapies could become a viable therapeutic option to slow disease progression in patients with T1D. Moreover, alternative methods aimed to mitigate excessive Th17 cell activity in patients with new-onset T1D warrant further investigation. For instance, one of the aforementioned methods may be the use of calcifediol (also known as 25-hydroxyvitamin D3 or calcidiol), which we recently described as a safe and effective adjuvant immunomodulatory agent able to sustain the clinical remission phase of T1D[94]. In particular, a 22-year-old man with new-onset T1D was prescribed calcifediol shortly after the disease diagnosis (oral administration at a dose of 0.266 mg every 20 days). He exhibited an unusually prolonged duration (31 months) of the clinical remission phase of T1D, which was accompanied by a low number of Th17 cells (as indicated by the intracellular expression of RORγt) identified through the flow cytometric immunophenotyping of peripheral blood lymphocytes performed at 24 mo from the disease diagnosis[94]. Accordingly, evidence suggests that vitamin D can exert a wide range of anti-inflammatory and immunomodulatory actions, including the inhibition of Th1 and Th17 cell development, as well as the stimulation of Treg differentiation and suppressive capacity[3].

A recent multicenter, double-blind, randomized phase 2 trial conducted on 72 adolescents aged 12-18 years with recent-onset T1D showed that treatment with ustekinumab (a fully human monoclonal antibody binding to the shared p40 subunit of IL-12 and IL-23, thus targeting the development of Th1 cells and Th17 cells) significantly preserved endogenous insulin secretion, as measured by stimulated plasma C-peptide levels at 12 mo[95]. Of note, preservation of C-peptide was correlated with the reduction of T helper cells co-secreting IL-17A and IFN-γ (Th17.1 cells). In particular, there was a decrease in a subset of Th17.1 cells co-expressing IL-2 and granulocyte-macrophage colony-stimulating factor (IL-2+ GM-CSF+ Th17.1 cells). Importantly, ustekinumab treatment was well tolerated and not associated with an increase in adverse events[95]. These findings support the existence of an activated subset of Th17.1 cells, which play a role in T1D pathophysiology and can be targeted to counteract the gradual decline in C-peptide levels in patients with new-onset T1D. A few clinical trials are currently ongoing to explore the use of Th17- and IL-17-targeted therapies in patients with new-onset T1D (Table 2).

Table 2 Summary of the main ongoing clinical studies of Th17- and interleukin-17-targeted therapies in patients with new-onset type 1 diabetes.

Study name	Study status and planned number of participants	Country	Study type	Study outcomes	ClinicalTrials.gov ID
Clinical Phase II/III Trial of Ustekinumab to Treat Type 1 Diabetes (UST1D2)	Recruiting; 66 adults	Canada	Randomized, placebo-controlled, double-blinded, multicenter phase II/II study conducted on 66 adult subjects (18-35 yr old) with recent-onset T1D. Ustekinumab is a fully human monoclonal antibody binding to the shared p40 subunit of IL-12 and IL-23, thus targeting the development of Th1 cells and Th17 cells. Planned study duration: Patients will be followed for 78 wk after the administration of the first ustekinumab dose. There will be a total of 10 study visits over the 78-wk period, three of which will be non-dosing and follow-up visits	Assessment of the efficacy of ustekinumab in counteracting the decline in mixed meal-stimulated C-peptide values in adult patients with recent-onset T1D	NCT03941132
Ixekizumab Diabetes Intervention Trial (I-DIT)	Recruiting; 127 patients	Sweden	Double-blind, placebo-controlled prospective phase 2 trial conducted on adult patients with newly diagnosed T1D. Planned study duration: 52 wk	Assessment of the efficacy of ixekizumab (an anti-IL-17A humanized monoclonal antibody) in counteracting the decline in mixed meal-stimulated C-peptide values in adult patients with newly diagnosed T1D	NCT04589325

T1D: Type 1 diabetes; IL: Interleukin; Th: T helper.

Clinical studies have demonstrated how immune interventions, either by suppressing the autoimmune responses or fostering immunological tolerance, can be effective in partially preserving residual beta-cell function in patients with recent-onset T1D[96-98]. However, it is still unclear whether Th17 cell-targeted therapies are superior to IL-17 neutralization therapy in patients with T1D. Moreover, preclinical evidence suggests that IL-17 neutralization can prevent the onset of autoimmune diabetes after the development of insulitis but not earlier[56].

Therefore, it is worth investigating interventions targeting Th17 cells or IL-17, in order to understand whether they can effectively prevent (or significantly delay) the clinical onset of disease or prolong the clinical remission phase in patients with stage 2 and stage 3 T1D, respectively. If Th17- and IL-17-targeted therapies prove safe and effective during the aforementioned stages of T1D, their use may also be investigated in patients with multiple islet autoantibody positivity associated with normoglycemia and absence of clinical symptoms (stage 1 T1D). Importantly, presymptomatic stages of T1D (stages 1 and 2) can easily be identified through measurement of circulating islet autoantibodies and markers of glucose homeostasis (fasting blood glucose values, and/or HbA1c, and/or post-OGTT blood glucose values). To date, the majority of screening programs aimed at identifying subjects at risk for T1D have targeted relatives of people affected by the disease to improve yield and feasibility[99]. However, about 90% of subjects who develop T1D do not have a positive family history[99]. Existing T1D population screening programs rely on the identification of circulating islet autoantibodies (such as IAA, GADA, IA-2A, and ZnT8A) and genetic determinants of T1D risk (high-risk HLA genotypes and other non-HLA susceptibility loci). Some of these programs are also combined with screening for celiac disease. Besides yielding relevant information on disease progression and approaches for timing of T1D screening in clinical settings[99], these programs may also be useful for identifying T1D patients who are eligible for studies investigating the use of Th17- and IL-17-targeted therapies at different stages of the disease.

Future perspectives regarding Th17- and IL-17-targeted therapies for T1D patients include a deeper elucidation of the mechanisms underlying the involvement of Th17 cells and IL-17 in T1D pathophysiology, as well as the identification of reliable biomarkers for patient stratification and prediction of therapeutic outcomes following Th17/IL-17-targeted interventions. Notably, it will be interesting to investigate the following hypotheses: (1) Th17 cells play a critical role in the early stages of T1D-related beta-cell destruction, and targeting them may significantly delay disease onset; (2) IL-17 modulation could reduce T1D-related inflammation within the pancreatic islets (insulitis), leading to a significant preservation of residual beta-cell function; and (3) Combining Th17- and IL-17-targeted therapies with other existing immunotherapeutic agents may enhance treatment efficacy while minimizing adverse drug reactions. Testing these hypotheses in well-designed preclinical and clinical studies will provide valuable insights for the potential development of novel Th17- and IL-17-targeted therapies as disease-modifying agents for T1D. While preclinical investigations and preliminary clinical studies have highlighted the potential of Th17- and IL-17-targeted therapies in managing autoimmune and inflammatory diseases, robust clinical data regarding T1D are still scarce. This paucity of evidence makes it difficult to evaluate the long-term safety and efficacy of Th17- and IL-17-targeted therapies even in distinct subgroups of T1D patients, such as in patients with different T1D endotypes [25]. Furthermore, practical challenges regarding patient selection, optimal drug dosing and adverse event monitoring need to be thoroughly addressed through well-designed clinical studies. Finally, immune profiling by flow cytometric immunophenotyping of peripheral blood lymphocytes (including Th17 cells) and analysis of IL-17 pathway may also offer further insights into the optimal timing of Th17- and IL-17-targeted therapies in patients with T1D.

CONCLUSION

A growing body of evidence from animal and human studies links T1D pathophysiology to Th17 cells and IL-17 immunity in the setting of altered Tregs function. Therefore, the crucial role of Th17 cells and IL-17 in T1D pathophysiology and immune-mediated pancreatic beta-cell destruction offers opportunities for investigation of novel Th17/IL-17 targeted interventions as disease-modifying therapies for T1D. Indeed, such therapies may mitigate or halt beta-cell autoimmunity in patients with presymptomatic and new-onset T1D, preserving residual beta-cell function and potentially delaying or preventing the onset of chronic complications of diabetes mellitus. Future perspectives regarding this research area include a deeper elucidation of the mechanisms underlying the involvement of Th17 cells and IL-17 in T1D pathophysiology, as well as the identification of reliable biomarkers for patient stratification and prediction of therapeutic outcomes following Th17- and IL-17-targeted interventions. Future prospective studies are undoubtedly required to comprehend the exact therapeutic role of Th17- and IL-17-targeted therapies in T1D patients, considering that such interventions may hold promise not only for the prevention and treatment of T1D, but also for the prevention and treatment of other autoimmune diseases in which Th17 cells and IL-17 are crucially implicated.