Interleukin-32 (IL-32), first identified as natural killer cell transcript 4 (NK4), is a (generally) proinflammatory cytokine that has gained attention for its potential role in various inflammatory and autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease (3). IL-32 has 35 known splice variants (https://useast.ensembl.org/Homo_sapiens/Gene/Splice?g=ENSG00000008517;r=16:3065297-3082192) of which 29 produce proteins, varying in length from 131 to 234 amino acids. All of these, except IL-32γ, lack a typical secretory peptide sequence, and indeed bear little structural resemblance to other cytokines (3, 4). Orthologues have been found in primates but not in rodents, whilst in other mammals, putative homologues have very low sequence alignment with human IL-32 (5). The lack of a rodent model has likely contributed to the lack of knowledge of IL-32; however, in vitro and in vivo human studies are driving our better understanding of IL-32 in both health and disease settings.

IL-32 is highly expressed in CD4⁺ T cell subsets including Tregs, Th1, Th17, Th17.1, Tfh and Th2 cells, as well as activated and memory CD8⁺ T cells, with lower expression in naïve T cells (3, 6). In many cases, the isoforms produced have not been assessed, although the majority of PBMC subsets appear to produce at least the α, β, γ and δ isoforms (reviewed in (3)). Recent work suggests that CD4⁺ T cells predominantly produce IL-32β and are the major source of IL-32β found in the serum (7). IL-32 is also highly expressed in NK cells; however, expression is generally low in both monocytes and naïve B cells (3). IL-32 is often observed as upregulated in disease states, including autoimmunity, in these cells (3, 6, 8, 9).

The expression of IL-32 is induced in T cells, other leukocytes and cells such as epithelial cells and fibroblasts by various proinflammatory cytokines including IL-1β, TNFα, IFNγ, IL-12, IL-18 and IL-23 (reviewed in (3)). In NK cells IL-2 is a strong inducer of IL-32 with a somewhat weaker effect in T cells (10). Recently, it has been shown that IL-32 (particularly IL-32β) is produced in response to IL-2 and secreted via membrane pores and exosomes in response to TCR stimulation (7). In T cells, IL-32 can be induced by in vitro stimulation of T cells using anti-CD3 antibodies or PMA and ionomycin (10). This induction by pro-inflammatory cytokines and T cell activation has important implications for the role of IL-32 in T1D.

IL-32 can also be induced in response to hypoxia via HIF1α and cysteamine dioxygenase (ADO) (11). As both IL-1β and IL-18 can induce IL-32, it is not surprising that innate immune receptors such as Toll-like receptors (TLRs) (12, 13) and inflammasomes e.g. NLPR3 (14), which induce IL-1β and IL-18, are associated with increased IL-32 induction. Recent research has drawn attention to both the role of hypoxia (15) and TLR signaling (16) in T1D development, suggesting an additional role for IL-32 in T1D pathogenesis.

A classical receptor for IL-32 has not been identified; however it can bind proteinase 3 (PR3), a neutrophil granule serine protease (17), and can bind integrins (αVβ3, αVβ6 but not αVβ8) through an RGD domain (18, 19). Although, as mentioned above, most isoforms lack a secretory domain, IL-32 can be measured in the serum and therefore is likely to function both intra- and extracellularly.

The most widely studied isoforms (α, β, γ, δ), are thought to have distinct biological functions (5) with all four inducing IL-6 production from PBMC, but only the latter three capable of inducing TNFα (20). The isoform-specific actions of IL-32 have been summarized previously (3). All IL-32 isoforms can induce IL-8 production, although IL-32γ and IL-32θ were the most potent (18). IL-32γ is thought to be more proinflammatory, inducing TNFα and IL-6 in rheumatoid arthritis synovial fibroblasts whilst the IL-32β isoform is thought to reduce inflammation (21). Pro-inflammatory cytokines such as TNFα and IFNγ are known to play important roles in T1D development and thus it can be seen that IL-32 may contribute to T1D pathology (1).

Whilst lentiviral knock-down of IL-32 expression reduced CD8⁺ T cell production of IFNγ, it also reduced expression of FoxP3 by Tregs in vitro (22). Conversely, in vitro culture of PBMCs with IL-32α led to Treg cell death and downregulation of FoxP3 expression (9). These apparently contradictory studies are likely explainable by the role of different IL-32 isoforms acting intracellularly or extracellularly. Therefore, care must therefore be taken when considering monitoring or targeting IL-32 therapeutically in T1D to analyze the various isoforms and assess their differential functions.

IL-32 can induce apoptosis in a wide variety of cell types including T cells (10). Again it is likely that this function is enacted by specific isoforms, as in cell lines IL-32γ and IL-32β, but not IL-32α, induced caspase-8-dependent cell death (23).

IL-32 can aid mitochondrial metabolism (via interactions with the electron transport chain and promotion of oxidative phosphorylation), and promotes proliferation, and differentiation of plasma cells (11) (this effect was driven by intracellular IL-32β and various IL-32 isoforms added to the extracellular media had no effect, highlighting the complexity of studying IL-32). As oxidative phosphorylation is also key in driving activation of T cells and decisions between pathogenic and regulatory T cells (24), the role IL-32 has in this setting should be further investigated.

IL-32, particularly the IL-32γ isoform, has been shown to activate Langerhans cells in the skin and induce CD80, HLA-DR and CXCL10 production (25). This is of particular interest in T1D, where CXCL10 is raised in the peripheral blood (26) and CXCR3 expressed in the islets aids recruitment of autoantigen-specific T cells and thus is a target for immunotherapy (27). In DCs, IL-32γ can upregulate the expression of the chemokines CCL2, CCL4, and CCL5, with upregulation of CCL5 in particular leading to increased chemotaxis of T cells (28). Further, IL-32 can induce monocytes to differentiate into macrophage-like cells (29) and IL-32θ in particular can induce monocytes to differentiate to inflammatory M1 macrophages that produce IL-1β, TNFα and inducible nitric oxide synthase (30). In T1D due to the high numbers of macrophages that infiltrate the islets (31) and their role in driving CD8⁺ T cell destruction of the β-cells (32), IL-32 could therefore be important in modulating these autoimmune responses.

Coxsackie B infection is hypothesized to contribute to T1D development (68) and a vaccine, PRV-101, for coxsackie B is in clinical development with a view to preventing T1D (69). Expression of IL-32 in pancreatic islets is increased by Coxsackie B infection (50). Furthermore, reporter cell lines infected with enterovirus strains isolated from Network for Pancreatic Organ Donors with Diabetes (nPOD) pancreases exhibited increased IL-32 expression compared to those infected with control enterovirus strains (64). Therefore, a potential mechanism of action of the vaccine may be through suppression of IL-32 production and thus should be investigated.

We have recently demonstrated that Ustekinumab, which blocks IL-12 and IL-23 signaling, can slow the loss of insulin production from β-cells in new-onset T1D (66). IL-32 is induced by IL-12 signaling in NK cells and by IL-12, IL-23, IL-2 and IFNγ signaling in T cells ((7) and reviewed (5)). Therefore, Ustekinumab’s blockade of IL-12 and IL-23 signaling, coupled with the downstream decrease in dual IL-17/IFNγ-secreting Th17.1 cells, (particularly those that co-express IL-2), may reduce β-cell loss partly through suppression of IL-32 induction.

TNFα and IL-32 have both been shown to induce each other in a positive feedback loop in arthritis in PBMCs, lymphoid tissue and DCs (62) again suggesting that the effects of Golimumab and other immunotherapies targeting TNFα to slow T1D progression may involve suppression of IL-32 (70, 71).

As discussed above, IL-2 is thought to induce IL-32 expression. However, treatment with low-dose IL-2 is thought to preserve β-cell function primarily through the specific expansion of Treg cells and reduction of IL-21-producing CD4⁺ T cells, without activating effector subsets of T cells and NK cells (63, 72). An examination of IL-32 expression in participants receiving low-dose IL-2 showed a significant upregulation of IL-32 a month after the final dose of IL-2 in CD56^bright NK cells after stimulation with PMA and ionomycin (63). This population of IL-32-expressing CD56^bright NK cells induced by low-dose IL-2 is thought to have immunoregulatory properties and therefore the impact of increased IL-32 expression is not clear but should be examined in future studies. If the IL-32 isoforms upregulated are predominantly pro-inflammatory (e.g. IL-32γ) this may reduce the immunoregulatory effect of the CD56^bright NK cells, whereas if the IL-32 isoforms have anti-inflammatory actions (e.g. IL-32β) this may potentiate the immunoregulatory properties of the CD56^bright NK cells (21, 63).

Inhibition of JAK1 can reverse T1D in NOD mice (73). In humans, the JAK1/2 inhibitor Baricitinib was successful in slowing progression of T1D in a Phase 2 trial [BANDIT (74)], this research is being continued in the recently launched JAKPOT T1D study (NCT05743244) and T1DPlus (ISRCTN45965456). IL-32 has been demonstrated to upregulate JAK1 expression and increase activation of the JAK1 signaling pathway (61), therefore inhibition with Baricitinib may act to reduce the effects of overexpression of IL-32 in T1D.

Teplizumab, an anti-CD3 monoclonal antibody, currently the sole licensed immunotherapy for delaying the onset of T1D in the USA (75) acts via inhibition of CD3 activation during TCR signaling, leading to modification of T cell subset abundance and the partial exhaustion of CD8⁺ T cells (65). In scRNAseq analysis, IL32 gene expression was significantly downregulated in CD4⁺ T cells from individuals treated with Teplizumab compared to placebo controls at 18 months (65).

Abatacept, a fusion protein of the extracellular domain of CTLA4 fused to the Fc region of IgG1,has been shown to decrease Th17 and Th1 cell abundance and proliferation in in rheumatoid arthritis (59, 60) and its efficacy is linked to baseline levels of Th17.1 cells. Therefore it may reduce IL-32 expression in T1D through decreased IFNγ signaling; however, as Abatacept also decreases abundance of Tregs the overall balance of its effect on IL-32 levels should also be determined (76).

Verapamil has been shown in the CLVer study to slow C-peptide loss in T1D (77), a result that is being followed up in Vera-T1D [awaiting publication of results (78)]. Verapamil will also be given to all participants in T1DPlus (in addition to immunotherapies in different arms of the trial). In human islet samples in vitro treatment with verapamil has been shown to reduce IL-32 expression and it is hypothesized that this is one of the main pathways through which it exerts its protective effects on β-cells (67). Further investigation is needed to assess the effect of verapamil on IL-32 in immune cells.