Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3′ UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai–Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-γ (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94–5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17–9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-γ (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3′ UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29–0.90; p = 0.020) and reduced circulating IFN-γ (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3′ UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-γ levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-γ levels.     

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.     

G(i)-protein-dependent inhibition of IL-12 production is mediated by activation of the phosphatidylinositol 3-kinase-protein 3 kinase B/Akt pathway and JNK

Ligands for certain G(i)-protein-coupled receptors (GiPCRs) potently inhibit the production of IL-12 by human monocytes. We addressed the intracellular signaling mechanisms by which this occurs using primary human cells. Stimulation with the GiPCR ligands C5a and 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purine-9-y1]-N-methyl-beta-D-ribofuranuronamide (IB-MECA) blocked the production of IL-12 p70 by human monocytes stimulated with LPS and IFN-gamma. In addition, C5a reduced the expression of mRNA for IL-12 p35, p40, IL-23 p19, and IL-27 p28. This effect was due neither to a down-regulation of TLR4 or IFN-gamma receptor on the cell surface nor to interference with IFN-gamma signaling, because IFN-gamma-induced up-regulation of HLA-DR and CD40 were unaffected. C5a or IB-MECA activated the PI3K/Akt signaling pathway and induced the phosphorylation of the MAPK p38, ERK, and JNK. Inhibition of the PI3K/Akt signaling pathway with wortmannin or an inhibitor of Akt activity, and inhibition of JNK but not ERK prevented IL-12 and IL-23 suppression by C5a. These data extend observations on IL-12 suppression by C5a to IL-23 and IL-27, and are the first to demonstrate the intracellular signaling events leading to IL-12 and IL-23 inhibition after GiPCR activation.     

Polymorphisms of interleukin-4, -10 and 12B genes and diabetic retinopathy

In diabetic retinopathy (DR) and other angiogenesis-associated diseases, increased levels of cytokines, inflammatory cells, and angiogenic factors are present. We investigated the hypothesis that rs2243250 polymorphism of the interleukin 4 (IL-4) gene or rs1800896 polymorphism of the interleukin 10 (IL-10) gene, and rs3212227 polymorphism of the 3’ untranslated region (3’ UTR) of the interleukin-12 p40 gene (IL12B) may be associated with the development of proliferative diabetic retinopathy (PDR) in Caucasians with type 2 diabetes (DM2). This cross sectional case — control study included 189 patients with PDR and 187 patients with type 2 diabetes without PDR. Polymorphisms rs1800896 of the IL-10 gene, rs2243250 of the IL-4 gene, and rs3212227 of IL12B gene were analyzed by ARMS -PCR and RFLP -PCR methods. Multivariate analysis demonstrated the GG genotype of the rs1800896 polymorphism of the IL-10 gene to be associated with increased risk for PDR (OR=1.99; 95% CI=1.11–3.57; P=0.02), whereas the TT genotype of the rs2243250 polymorphism of the IL-4 gene and the AA genotype of the rs3212227 polymorphism of the IL-12 gene were not independent risk factors for PDR. Our findings suggest that the genetic variations at the IL-10 promoter gene might be a genetic risk factor for PDR in Caucasians with type 2 diabetes.     

The ratio of P40 monomer to dimer is an important determinant of IL-12 bioactivity

IL-12 is a 75 kDa heterodimer (IL12p70) comprised of independently regulated disulfide-linked 40 kDa (p40) and 35 kDa (p35) subunits. The p40 subunit exists extracellularly as a monomer (IL12p40) or dimer (IL12(p40)2) and can antagonize the action of IL12p70. Given the disagreement in the literature over the physiologic roles for IL12p70, IL12p40, and IL12(p40)2, we asked whether the bioactivity of IL-12 depended only on the concentration of the IL12p70 subunit alone or whether the relative concentrations of IL12p70, IL12p40, and IL12(p40)2 and their competitive binding with the IL-12 receptor are essential for determining IL-12 bioactivity under simulated human physiologic conditions. A mathematical model for IL-12 bioactivity was created by incorporating the production of IL12p70, IL12p40, and IL12(p40)2 by mature human DC and the interaction of these species with the IL-12 receptor. Using this model, we explored the effects of IFN-gamma, IL-4, and PGE2 concentrations on the bioactivity of IL-12. The simulations suggest that the concentration of IL12p70 alone is not indicative of IL-12 bioactivity; rather, the bioactivity of IL-12 produced by mature DC depends on IL12p70, IL12p40, and IL12(p40)2 production and their competitive interaction with the IL-12 receptor. In addition to the typically measured quantities of total p40 (IL12p40 + IL12(p40)2) and IL12p70, the ratio of IL12p40 to IL12(p40)2 is an equally important, yet underreported, determinant of IL-12 bioactivity.     

Impaired production of IL-12 in systemic lupus erythematosus. III: deficient IL-12 p40 gene expression and cross-regulation of IL-12, IL-10 and IFN-gamma gene expression.

Interleukin 12 (IL-12) is a heterodimer comprising p35 and p40 subunits which are encoded and regulated separately. The authors previously demonstrated deficient IL-12 production in SLE which correlates negatively with disease activity. The present study was designed to determine whether deficiency of IL-12 and excess production of IL-10 and IL-6 in systemic lupus erythematosus (SLE) are due to aberrant regulation at the gene level. Using semiquantitative RT-PCR assay, it was shown that constitutive expression of IL-12 p35 gene is somewhat impaired in SLE compared with controls and that IL-12 p40 mRNA, which was present at low levels in controls, was undetectable in unstimulated SLE peripheral blood mononuclear cells (PBMC). Gene expression of IL-12 p35 and p40 was significantly increased in response to SAC, with significantly lower SAC-induced expression of p40 in SLE patients than controls. SAC-stimulated IL-12 p35 and p40 mRNAs were significantly augmented by interferon gamma (IFN-gamma). Exogenous IL-12 or IFN-gamma significantly inhibited IL-10 gene expression, without affecting IL-6 mRNA or other proinflammatory cytokine mRNA levels. These observations were further confirmed by studies of protein production at the single cell level using ELISPOT assay. Downregulation of IL-12 p40 expression appears to be the cause of IL12 p70 deficiency in SLE. If this defect could be repaired, normalization of IL-12 and IFN-gamma production should reduce excessive IL-10 and prevent pathology.     

Rac1 negatively regulates lipopolysaccharide-induced IL-23 p19 expression in human macrophages and dendritic cells and NF-kappaB p65 trans activation plays a novel role

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and of a p40 subunit that is also common to IL-12. We defined the distinct signaling mechanisms that regulate the LPS-mediated induction of IL-23 p19 and p40 in human macrophages and dendritic cells. We found that the overexpression of dominant-negative Rac1 (N17Rac1) enhanced LPS-induced IL-23 p19 expression but did not alter p40 expression or IL-12 p70 production in PMA-treated THP-1 macrophages and in human monocyte-derived dendritic cells. Although the inhibition of either p38 MAPK or JNK enhanced LPS-induced p19 expression, N17Rac1 did not influence either p38 MAPK or JNK activation. By contrast, N17Rac1 augmented both NF-kappaB gene expression and p65 trans activation stimulated by LPS without affecting the degradation of IkappaB-alpha or DNA binding to NF-kappaB. Furthermore, small interference RNA of NF-kappaB p65 attenuated cellular amounts of p65 and suppressed LPS-induced p19 expression but did not affect p40 expression. Our findings indicate that Rac1 negatively controls LPS-induced IL-23 p19 expression through an NF-kappaB p65 trans activation-dependent, IkappaB-independent pathway and that NF-kappaB p65 regulates LPS-induced IL-23 p19, but not p40, expression, which causes differences in the control of IL-23 p19 and p40 expression by Rac1.     

Association of IL-12, IL-18 variants and serum IL-18 with bladder cancer susceptibility in North Indian population

IL-12 and IL-18 are immunomodulatory cytokines that play important roles in host immune response against cancers. Variation in DNA sequence in gene promoter may lead to altered IL-18 production and/or activity, and hence can modulate an individual's susceptibility to BC. To test this hypothesis, we investigated the relationship of IL-18 gene promoter −137 G/C and −607C/A polymorphisms and IL12 (− 16974) A/C with the risk of BC in North Indian population. Polymorphisms in IL-18 and IL-12 genes were analyzed in 200 BC patients and 200 age, ethnicity and sex-matched controls, using restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP) and amplification refractory mutation specific-polymerase chain reaction (ARMS) method. The concentrations of IL-18 in serum were determined by ELISA. Significant association was observed with IL18 (− 137) G/C heterozygous genotype (GC) with 1.96 folds risk of BC as well at C allele carrier and variant C allele having 2 fold and 1.6 fold risk for BC respectively. IL18 (− 607) C/A, heterozygous CA genotype also showed a high risk (OR = 1.59) for BC. While IL12 (− 16974) A/C heterozygote genotype and C allele carrier demonstrated reduced risk of BC. Hetero genotype of IL18 (− 137) G/C was associated with risk of recurrence (HR = 2.35) in superficial BC patients receiving BCG treatment thus showing least survival. The distributions of IL-18 gene haplotypes were not significantly different between patients and controls. Serum IL-18 levels were significantly higher in BC patients than in the healthy subjects (p = 0.025). Serum IL-18 levels was also significantly associated with IL18 (− 137) G/C in heterozygous genotype (GC) (p = 0.048). Our results suggest that IL-18 gene polymorphism contributes to bladder cancer risk whereas IL-12 is protective. A relation between IL18 (− 137) G/C in heterozygous genotype with elevated IL-18 serum level and bladder cancer risk has been registered in the present study.     

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL-12p70, through which they play a direct role in the development of IFN-gamma-secreting Th1 cells, costimulation of CTL differentiation and NK-cell activation. In contrast, IL-10, which is also produced by DC, negatively regulates IL-12 production. IL-12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL-12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL-12p70 secretion by monocyte-derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL-12p70, presence of the IL10 high genotype restricted IL-12p70 production in these individuals. These observations provide a further genetic control of IL-12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.     

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.     

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.     

Soluble bacterial constituents down-regulate secretion of IL-12 in response to intact Gram-positive bacteria

Intact Gram-positive bacteria induce production of large amounts of IL-12 from freshly isolated human monocytes. Here the bacterial structures and signalling pathways involved were studied and compared with those leading to IL-6 production, and to IL-12 production in response to LPS after IFN-gamma pre-treatment. Intact bifidobacteria induced massive production of IL-12 (1 ng/ml) and IL-6 (>30 ng/ml) from human PBMC, whereas fragmented bifidobacteria induced IL-6, but no IL-12. IL-12 production induced by intact bifidobacteria was inhibited by pre-treatment with bifidobacterial sonicate, peptidoglycan, muramyl dipeptide, lipoteichoic acid, the soluble TLR2 agonist Pam(3)Cys-SK(4), or anti-TLR2 antibodies. Blocking of phagocytosis by cytochalasin, inhibition of the JNK or NF-kappaB pathways or treatment with Wortmannin also reduced the IL-12 response to intact Gram-positive bacteria. LPS induced moderate levels of IL-12 (0.31 ng/ml), but only from IFN-gamma pre-treated PBMC. This IL-12 production was enhanced by Wortmannin and unaffected by blocking the JNK pathway. Thus, intact Gram-positive bacteria trigger monocyte production of large amounts of IL-12 via a distinct pathway that is turned off by fragmented Gram-positive bacteria. This may be a physiological feedback, since such fragments may signal that further activation of the phagocyte via the IL-12/IFN-gamma loop is unnecessary.