Recombinant p35 from Bacteria Can Form Interleukin (IL-)12, but Not IL-35

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40_C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40_C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35_bac). Reconstitution of IL-35 with p35_bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.     

Astrocytes as antigen-presenting cells: expression of IL-12/IL-23

Interleukin-12 (IL-12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1-type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL-23, composed of the same p40 subunit as IL-12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen-presenting cell (APC) function can present antigen to myelin basic protein (MBP)-reactive T cells, and whether this presentation is blocked with antibodies against IL-12/IL-23p40. Interferon (IFN)-gamma-treated APC induced proliferation of MBP-reactive T cells. Anti-IL-12/IL-23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL-12/IL-23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL-12p70. Because IL-12 and IL-23 share p40, we wanted to determine whether astrocytes also express IL-12p35 and IL-23p19, as microglia were already shown to express them. Astrocytes expressed IL-12p35 mRNA constitutively, and IL-23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL-12/IL-23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti-IL-12/IL-23p40 antibodies.     

Contribution of IL-12/IL-35 Common Subunit p35 to Maintaining the Testicular Immune Privilege

The testis is an organ with immune privilege. The comprehensive blood–testis barrier formed by Sertoli cells protects autoimmunogenic spermatozoa and spermatids from attack by the body’s immune system. The interleukin (IL)-6/IL-12 family cytokines IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Epstein-Barr virus−induced gene 3 [EBI3]), and IL-35 (p35/EBI3) play critical roles in the regulation of various immune responses, but their roles in testicular immune privilege are not well understood. In the present study, we investigated whether these cytokines are expressed in the testes and whether they function in the testicular immune privilege by using mice deficient in their subunits. Expression of EBI3 was markedly increased at both mRNA and protein levels in the testes of 10- or 12-week-old wild-type mice as compared with levels in 2-week-old mice, whereas the mRNA expression of p40 was markedly decreased and that of p35 was conserved between these two groups. Lack of EBI3, p35, and IL-12 receptor β2 caused enhanced infiltration of lymphocytes into the testicular interstitium, with increased interferon-γ expression in the testes and autoantibody production against mainly acrosomal regions of spermatids. Spermatogenic disturbance was more frequently observed in the seminiferous tubules, especially when surrounded by infiltrating lymphocytes, of these deficient mice than in those of wild-type mice. In particular, p35-deficient mice showed the most severe spermatogenic disturbance. Immunohistochemical analyses revealed that endothelial cells and peritubular cells in the interstitium were highly positive for p35 at both ages, and CD163⁺ resident macrophages positive for p35 and EBI3, possibly producing IL-35, were also detected in the interstitium of 12-week-old mice but not those of 2-week-old mice. These results suggest that p35 helps in maintaining the testicular immune privilege, in part in an IL-35-dependent manner.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.     

The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12

Interleukin (IL)-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an α-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3-Å crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a ‘tilt’ and ‘roll’ that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of a pocket on p40. This ‘arginine pocket’ is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes ‘mimic’ the network of interactions constituting the central arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pairwise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.     

Structural Basis for the Dual Recognition of IL-12 and IL-23 by Ustekinumab

Interleukin (IL)-12 and IL-23 are heterodimeric proinflammatory cytokines that share a common p40 subunit, paired with p35 and p19 subunits, respectively. They represent an attractive class of therapeutic targets for the treatment of psoriasis and other immune-mediated diseases. Ustekinumab is a fully human monoclonal antibody (mAb) that binds specifically to IL-12/IL-23p40 and neutralizes human IL-12 and IL-23 bioactivity. The crystal structure of ustekinumab Fab (antigen binding fragment of mAb), in complex with human IL-12, has been determined by X-ray crystallography at 3.0 Å resolution. Ustekinumab Fab binds the D1 domain of the p40 subunit in a 1:1 ratio in the crystal, consistent with a 2 cytokines:1 mAb stoichiometry, as measured by isothermal titration calorimetry. The structure indicates that ustekinumab binds to the same epitope on p40 in both IL-12 and IL-23 with identical interactions. Mutational analyses confirm that several residues identified in the IL-12/IL-23p40 epitope provide important molecular binding interactions with ustekinumab. The electrostatic complementarity between the mAb antigen binding site and the p40 D1 domain epitope appears to play a key role in antibody/antigen recognition specificity. Interestingly, this structure also reveals significant structural differences in the p35 subunit and p35/p40 interface, compared with the published crystal structure of human IL-12, suggesting unusual and potentially functionally relevant structural flexibility of p35, as well as p40/p35 recognition. Collectively, these data describe unique observations about IL-12p35 and ustekinumab interactions with p40 that account for its dual binding and neutralization of IL-12 and IL-23.     

IL-12Rbeta2-deficient mice of a genetically resistant background are susceptible to Leishmania major infection and develop a parasite-specific Th2 immune response

The cytokine IL-12 plays a critical role in inducing the production of IFN-gamma from T and NK cells and in the polarization of T cells towards the Th1 phenotype. IL-12 is comprised of two subunits (IL-12p40 and IL-12p35) that together form the biologically active p70 molecule, and IL-12 functions via binding to a heterodimeric receptor (IL-12Rbeta1 and IL-12Rbeta2). Previous studies utilizing mice deficient for either the IL-12 cytokine or the IL-12-induced signaling molecule STAT4 have established a critical role for IL-12 during infection with Leishmania major. However, these studies warrant careful re-interpretation in light of the recent discovery of the IL-12-related cytokine, IL-23, which utilizes the IL-12p40 chain in combination with an IL-12p35-related molecule, called p19, and a receptor comprised of the IL-12Rbeta1 chain plus a unique chain referred to as IL-23R. We analyzed the course of L. major infection in mice deficient for the IL-12-specific IL-12Rbeta2 subunit in order to assess the role of IL-12 signaling without disruption of the IL-23 pathway. After infection with L. major, IL-12Rbeta2KO mice of a resistant background (C57Bl/6) developed large cutaneous lesions similar to those developed by susceptible BALB/c mice. Draining lymph node cells from L. major-infected IL-12Rbeta2KO mice released the Th2 cytokines IL-4 and IL-5 after in vitro stimulation with Leishmania lysate but were completely devoid of IFN-gamma, consistent with a default towards a strong parasite-specific Th2 response. L. major-infected IL-12Rbeta2KO mice were also devoid of parasite-specific IgG2a antibodies, and interestingly, their footpad lesions ulcerated earlier than those of susceptible BALB/c mice.     

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.     

An Interspecies Analysis Reveals Molecular Construction Principles of Interleukin 27

Interleukin 27 (IL-27) is a cytokine that regulates inflammatory responses. It is composed of an α subunit (IL-27α) and a β subunit (EBI3), which together form heterodimeric IL-27. Despite this general principle, IL-27 from different species shows distinct characteristics: Human IL-27α is not secreted autonomously while EBI3 is. In mice, the subunits show a reciprocal behavior. The molecular basis and the evolutionary conservation of these differences have remained unclear. They are biologically important, however, since secreted IL-27 subunits can act as cytokines on their own.Here, we show that formation of a single disulfide bond is an evolutionary conserved trait, which determines secretion-competency of IL-27α. Furthermore, combining cell-biological with computational approaches, we provide detailed structural insights into IL-27 heterodimerization and find that it relies on a conserved interface. Lastly, our study reveals a hitherto unknown construction principle of IL-27: one secretion-competent subunit generally pairs with one that depends on the other to induce its secretion.Taken together, these findings significantly extend our understanding of IL-27 biogenesis as a key cytokine and highlight how protein assembly can influence immunoregulation.     

Peroxisome proliferator-activated receptor-alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis

The interleukin-12 (IL-12) family of cytokines which includes IL-12, IL-23, and IL-27 play critical roles in T cell differentiation and are important modulators of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Previously, we demonstrated that peroxisome proliferator-activated receptor (PPAR) -α agonists suppress the development of EAE. The present studies demonstrated that the PPAR-α agonist fenofibrate inhibited the secretion of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 by lipopolysaccharide-stimulated microglia. The cytokines interferon-γ and tumor necrosis factor-α also stimulated IL-12 p40 and IL-27 p28 expression by microglia, which was suppressed by fenofibrate. Furthermore, fenofibrate inhibited microglial expression of CD14 which plays a critical role in TLR signaling, suggesting a mechanism by which this PPAR-α agonist regulates the production of these pro-inflammatory molecules. In addition, fenofibrate suppressed the secretion of IL-12p40, IL-23, and IL-27p28 by lipopolysaccharide-stimulated astrocytes. Importantly, fenofibrate suppression of EAE was associated with decreased expression of IL-12 family cytokine mRNAs as well as mRNAs encoding TLR4, CD14, and MyD88 known to play critical roles in MyD88-dependent TLR signaling. These novel observations suggest that PPAR-α agonists including fenofibrate may modulate the development of EAE, at least in part, by suppressing the production of IL-12 family cytokines and MyD88-dependent signaling.     

Peroxisome proliferator-activated receptor-gamma agonists suppress the production of IL-12 family cytokines by activated glia

The IL-12 family of cytokines, which include IL-12, IL-23, and IL-27, play critical roles in the differentiation of Th1 cells and are believed to contribute to the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Relatively little is known concerning the expression of IL-12 family cytokines by cells of the CNS, the affected tissue in MS. Previously, we and others demonstrated that peroxisome proliferator-activated receptor (PPAR)-gamma agonists suppress the development of EAE, alter T cell proliferation and phenotype, and suppress the activation of APCs. The present studies demonstrated that PPAR-gamma agonists, including the naturally occurring 15-deoxy-Delta(12,14)-PGJ(2) and the synthetic thiazoladinedione rosiglitazone, inhibited the induction of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 proteins by LPS-stimulated primary microglia. In primary astrocytes, LPS induced the production of IL-12p40, IL-23, and IL-27p28 proteins. However, IL-12p70 production was not detected in these cells. The 15-deoxy-Delta(12,14)-PGJ(2) potently suppressed IL-12p40, IL-23, and IL-27p28 production by primary astrocytes, whereas rosiglitazone suppressed IL-23 and IL-27p28, but not IL-12p40 in these cells. These novel observations suggest that PPAR-gamma agonists modulate the development of EAE, at least in part, by inhibiting the production of IL-12 family cytokines by CNS glia. In addition, we demonstrate that PPAR-gamma agonists inhibit TLR2, MyD88, and CD14 expression in glia, suggesting a possible mechanism by which these agonists modulate IL-12 family cytokine expression. Collectively, these studies suggest that PPAR-gamma agonists may be beneficial in the treatment of MS.     

Ovine interleukin 12 has biological activity on ovine and human activated peripheral blood mononuclear cells

Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits that form the biologically active p70 molecule, and is a potent inducer of the pro-inflammatory cytokine IFN-gamma. In this study the coding sequence for ovine interleukin 12 p35 and p40 subunits was derived by RT-PCR cloning. Ovine p35 and p40 cDNA sequences show a high level of similarity at the nucleic acid and protein levels when compared to corresponding bovine and human sequences. In particular, cysteine residues and N-linked glycosylation sites are conserved between species. Secretion of the IL-12 heterodimer from CHO cells co-transfected with ovine p35 and p40 cDNA was shown by immunoprecipitation of a 60 and 66 kDa protein from transfectant supernatant. In addition, the supernatant from co-transfected cells augmented the proliferation of Con A-activated ovine peripheral blood mononuclear cells (PBMNC). Cross-species activity was shown by the enhancement of proliferation of human phytohaemagglutinin (PHA)-activated PBMNC. Supernatants from co-transfectants of hu p35/ov p40 and ov p35/hu p40, to generate chimeric heterodimers, also demonstrated stimulatory activity. Human and chimeric IL-12-induced proliferation of activated PBMNC was inhibited using an anti-human IL-12 polyclonal antibody, however this antibody showed minimal inhibition of ovine IL-12. This study suggests that ovine IL-12 has biological properties similar to its human counterpart.     

IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12

IL-23, a heterodimeric cytokine composed of the p40 subunit of IL-12 and a novel p19 subunit, has been shown to be a key player in models of autoimmune chronic inflammation. To investigate the role of IL-23 in host resistance during chronic fungal infection, wild-type, IL-12- (IL-12p35-/-), IL-23- (IL-23p19-/-), and IL-12/IL-23- (p40-deficient) deficient mice on a C57BL/6 background were infected with Cryptococcus neoformans. Following infection, p40-deficient mice demonstrated higher mortality than IL-12p35-/- mice. Reconstitution of p40-deficient mice with rIL-23 prolonged their survival to levels similar to IL-12p35-/- mice. IL-23p19-/- mice showed a moderately reduced survival time and delayed fungal clearance in the liver. Although IFN-gamma production was similar in wild-type and IL-23p19-/- mice, production of IL-17 was strongly impaired in the latter. IL-23p19-/- mice produced fewer hepatic granulomata relative to organ burden and showed defective recruitment of mononuclear cells to the brain. Moreover, activation of microglia cells and expression of IL-1beta, IL-6, and MCP-1 in the brain was impaired. These results show that IL-23 complements the more dominant role of IL-12 in protection against a chronic fungal infection by an enhanced inflammatory cell response and distinct cytokine regulation.     

Suppression of ongoing adjuvant-induced arthritis by neutralizing the function of the p28 subunit of IL-27

IL-27 is a recently defined family member of the long-chain four-helix bundle cytokines, which consists of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of inflammatory autoimmune diseases has never been studied. The current study uses the DNA vaccination technology, and highly specific Abs to the p28 subunit of IL-27 that were generated by this technology, to delineate its role in the regulation of adjuvant-induced arthritis in Lewis rats. Neutralizing the in vivo function of IL-27 by targeted DNA vaccines and by Abs against IL-27 p28 that were produced in protected donors could rapidly suppress an ongoing disease. Disease suppression was associated with a reduced ex vivo production of inflammatory cytokines. We then used these Abs to investigate the mechanistic basis of disease suppression, showing that IL-27 is not only involved in directing the polarization of naive T cells, but also affects the proliferative response and cytokine production of Ag-specific effector/memory Th1 cells. This may explain, in part, its important role in the regulation of inflammatory autoimmune diseases, and also suggest novel ways of therapy.     

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 Å, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.