Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

Isochromans and Related Constituents from the Endophytic Fungus Annulohypoxylon truncatum of Zizania caduciflora and Their Anti-Inflammatory Effects.

Six new isochroman derivatives (annulohypoxylomans A-C, 1-3; annulohypoxylomanols A and B, 6 and 7; and annulohypoxyloside, 8), an isocoumarin (annulohypoxylomarin A, 4), and an azaphilone derivative (xylariphilone, 5) were isolated from an ethyl acetate extract derived from cultures of the endophytic fungus JS540 found in the leaves of Zizania caduciflora. The JS540 strain was identified as Annulohypoxylon truncatum. The structures of the isolated compounds were elucidated by one- and two-dimensional nuclear magnetic resonance and mass spectrometry and by comparison with related compounds from the literature. The anti-inflammatory activities of the isolated compounds were evaluated in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells. Xylariphilone (5) exhibited significant inhibitory effects on LPS-induced interleukin (IL)-6, IL-12 p40, and tumor necrosis factor (TNF)-α production with IC50 values of 5.3, 19.4, and 37.6 µM, respectively.

Pretreatment with Antiviral Preparation Kagocel Normalizes the Content of Bone Marrow Multipotent Stromal Cells and TNFα in Blood Serum of CBA Mice Disturbed by Administration of S. typhimurium Antigen Complex In Vivo and Maintains High Concentration of IL-10 and Th1 Cytokines.

Pretreatment with the active substance of antiviral preparation Kagocel, inductor of type I endogenous IFN, in a daily therapeutic dose (30 µg/mouse) 3 h prior to administration of S. typhimurium antigens to CBA mice reduced the number of bone marrow multipotent stromal cell (significantly increased by 3.2 times on the next day after antigen injection) to the initial level. Thus, activation of the stromal tissue induced by administration of the bacterial antigen was blocked. In addition, preliminary administration of Kagocel modulated the cytokine profile of the blood serum affected by S. typhimurium antigens: reduced 1.6-fold elevated concentration a proinflammatory cytokine TNFα to the control level (in 4 h after antigen injection) and maintained this level in 20 h after antigen administration. Kagocel also maintained the concentration of anti-inflammatory cytokine IL-10 at the level surpassing the normal by 1.6 times and high concentrations of Th1 cytokines (IL-2, IFNγ, and IL-12). These results suggest that Kagocel can reduce the immune response to bacterial antigens (similar to type I IFN [7]), which can contribute to its therapeutic and preventive effects in addition to its well documented antiviral activity and then this preparation can be used for the therapy of diseases accompanied by excessive or chronic inflammation.

RCAI-61 and related 6'-modified analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce interferon-γ in vivo.

We synthesized ten new analogs of 6'-modified KRN7000 (A): RCAI-58, 61, 64, 83, 85-87, 113, 119, and 125. They could be synthesized by α-selective galactosylation of ceramide 9 with the 6-modified D-galactopyranosyl fluorides (8a-8f) or L-arabinopyranosyl fluoride (17), or by etherification of the known alcohol 19. Bioassay of the ten analogs demonstrated that RCAI-61 (1, 6'-O-methylated analog of A) was the most potent immunostimulant among them, and could induce the production of a large amount of IFN-γ even at a low concentration in mice in vivo.

IFN-γ and IL-12 synergize to convert in vivo generated Th17 into Th1/Th17 cells.

Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for re-expression of IFN-γ and IL-17, by upregulated expression of T-bet and retinoic acid-related orphan receptor γt (RORγt), respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN-γ and IL-17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF-ß/IL-6 and IL-12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN-γ and IL-12 signaling. IFN-γ is required to upregulate expression of the IL-12Rß2 chain, and IL-12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORγt and T-bet at the single-cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro-inflammatory potential of Th1 and Th17 cells.

IL-18 inhibits TNF-alpha-induced osteoclastogenesis possibly via a T cell-independent mechanism in synergy with IL-12 in vivo.

It has recently been reported that tumor necrosis factor (TNF)-alpha has the ability to accelerate osteoclastogenesis. We previously reported that the proinflammatory cytokine interleukin (IL)-18 inhibits TNF-alpha-mediated osteoclastogenesis in mouse bone marrow cultures. In the present study, the effect of IL-18 on TNF-alpha-mediated osteoclastogenesis was investigated in vivo. We administered TNF-alpha with or without IL-18 into the supracalvaria of mice. The number of osteoclasts in the suture of the calvaria was increased in mice administered TNF-alpha. The number of osteoclasts in mice administered both TNF-alpha and IL-18 was lower than that in mice administered TNF-alpha alone. We previously showed that IL-12 and IL-18 synergistically inhibit TNF-alpha-mediated osteoclastogenesis in vitro. To assess the ability of these two cytokines to synergistically inhibit TNF-alpha-induced osteoclastogenesis in vivo, mice were administered the two cytokines at doses that did not inhibit osteoclast formation. The combination of IL-12 and IL-18 markedly inhibited TNF-alpha-induced osteoclastogenesis in vivo. To evaluate how IL-12 and IL-18 synergistically affect TNF-alpha-induced osteoclastogenesis, the IL-18 receptor (IL-18R) and IL-12R expression levels were analyzed by RT-PCR in bone marrow cells cultured with IL-12 or IL-18. IL-18R mRNA was increased in cells cultured with IL-12, while IL-12R mRNA was increased in cells cultured with IL-18. In addition, IL-18 inhibited TNF-alpha-induced osteoclastogenesis in mice with T-cell depletion caused by anti-CD4 and anti-CD8 antibodies. The present results suggest that IL-18 may inhibit TNF-alpha-mediated osteoclastogenesis in vivo via a T cell-independent mechanism.

Selected commensal-related bacteria and Toll-like receptor 3 agonist combinatorial codes synergistically induce interleukin-12 production by dendritic cells to trigger a T helper type 1 polarizing programme.

Enteric infections remain a major health problem causing millions of deaths in developing countries. The interplay among the host intestinal epithelium, the mucosa-associated immune system and microbiota performs an essential role in gut homeostasis and protection against infectious diseases. Dendritic cells (DCs) play a key role in orchestrating protective immunity and tolerance in the gut. The mechanisms by which DCs adapt their responses and discriminate between virulent microbes and trillions of innocuous bacteria remain ill-defined. Here we investigated the effect of cross-talk between commensal-related bacteria (CB) and Toll-like receptor (TLR) agonists on DC activation and the outcome of the in vitro T helper response. Human monocyte-derived DCs were exposed to eight different Gram-positive or Gram-negative CB strains prior to activation with five different TLR agonists. The key polarizing cytokines interleukin (IL)-12p70, IL-10, IL-1beta and IL-6 were quantified and the fate of naïve T-cell differentiation was evaluated. We identified a unique combination of Lactobacillus casei and TLR3 signals that acted in synergy to selectively increase IL-12p70 secretion. Exposure to poly(I:C) converted L. casei-treated DCs into potent promoters of T helper type 1 (Th1) responses. We propose that DCs can integrate harmless and dangerous non-self signals delivered by viral products, to mount robust Th1 responses. Thus, in vivo DC targeting with selective probiotics may improve strategies for the management of enteric diseases.

Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier.

What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRL(lpr/lpr) mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)-6, IL-12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non-nephritic mice. This was associated with down-regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRL(lpr/lpr) mice. Bacterial lipopeptide activates Toll-like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)-gamma induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRL(lpr/lpr) mice.

Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists.

Tumor-targeted immunomodulation using oncolytic viral vectors is currently being investigated as a promising strategy in cancer therapy. In a previous study, we showed that a measles virus Schwarz vaccine strain (MeVac) vector encoding an interleukin-12 fusion protein (FmIL-12) is an effective immunotherapy in the MC38cea murine colon adenocarcinoma model. We hypothesized that MeVac encoding interleukin-15 may mediate enhanced T and NK cell responses and thus increase the therapeutic efficacy, especially in NK cell-controlled tumors. Therefore, we generated MeVac vectors encoding an interleukin-15 superagonist, FmIL-15. Replication and oncolytic capacity, transgene expression, and functionality of MeVac FmIL-15 vectors were validated in vitro. Effects on the tumor immune landscape and therapeutic efficacy of both FmIL-12 and FmIL-15 vectors were studied in the MC38cea and B16hCD46 tumor models. Treatment with MeVac FmIL-15 increased T and NK cell infiltration in both models. However, MeVac FmIL-12 showed more robust viral gene expression and immune activation, resulting in superior anti-tumor efficacy. Based on these results, MeVac encoding a human IL-12 fusion protein was developed for future clinical translation.

Monocyte-derived interleukin-10 depresses the Bordetella pertussis- specific gamma interferon response in vaccinated infants.

Antigen-specific gamma interferon (IFN-gamma) has been demonstrated to participate in protection against Bordetella pertussis infection. Circulating mononuclear cells from B. pertussis-infected and from pertussis-vaccinated infants secrete high amounts of IFN-gamma after in vitro stimulation by B. pertussis antigens, but with a large variation in the secreted IFN-gamma levels between individuals. We show here that the inhibition of the specific IFN-gamma response can be at least partially attributed to IL-10 secretion by monocytes. This IL-10 secretion was not associated with polymorphisms at positions -1082, -819, and -592 of the IL-10 gene promoter, suggesting that other genetic or environmental factors affect IL-10 expression and secretion.

Systemic dendritic cell mobilization associated with administration of FLT3 ligand to SIV- and SHIV-infected macaques.

Reports indicate that myeloid and plasmacytoid dendritic cells (mDCs and pDCs), which are key effector cells in host innate immune responses, can be infected with HIV-1 and are reduced in number and function during the chronic phase of HIV disease. Furthermore, it was recently demonstrated that a sustained loss of mDCs and pDCs occurs in SIV-infected macaques. Since loss of functional DC populations might impair innate immune responses to opportunistic microorganisms and neoplastic cells, we explored whether inoculation of naive and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-ligand (FLT3-L) would expand the number of mDCs and pDCs in vivo. After the macaques received supraphysiologic doses of FLT3-L, mDCs, pDCs, and monocytes increased up to 45-fold in blood, lymph nodes, and bone marrow (BM), with DC expansion in the BM preceding mobilization in blood and lymphoid tissues. FLT3-L also increased serum levels of IL-12, at least transiently, and elicited higher surface expression of HLA-DR and the activation markers CD25 and CD69 on NK and T cells. During and after treatment of infected animals, APCs increased in number and were activated; however, CD4(+) T cell numbers, virion RNA, and anti-SIV/SHIV antibody titers remained relatively stable, suggesting that FLT3-L might be a safe modality to expand DC populations and provide therapeutic benefit during chronic lentivirus infections.

The shiitake mushroom-derived immuno-stimulant lentinan protects against murine malaria blood-stage infection by evoking adaptive immune-responses.

Lentinan, a (1-3)-beta glucan from Lentinus edodes, is an effective immunostimulatory drug. We tested the effects of lentinan during blood-stage infection by Plasmodium yoelii 17XL (P.y17XL). Pre-treatment of mice with lentinan significantly decreased the parasitemia and increased their survival after infection. Enhanced IL-12, IFN-gamma and NO production induced by lentinan in spleen cells of infected mice revealed that the Th1 immune response was stimulated against malaria infection. In vitro and in vivo, lentinan can result in enhanced expression of MHC II, CD80/CD86, and Toll-like receptors (TLR2/TLR4), and increased production of IL-12 in spleen dendritic cells (DCs) co-cultured with parasitized red blood cells (pRBCs). Moreover, both the number of CD4(+)CD25(+) regulatory T cells (Tregs) and the levels of IL-10 secreted by Tregs were reduced by pre-treatment with lentinan in the spleen of malaria-infected mice. Meanwhile, apoptosis of CD4(+) T cell in spleens of mice pretreated with lentinan was significantly reduced. In summary, lentinan can induce protective Th1 immune responses to control the proliferation of malaria parasites during the blood-stage of P.y17XL infection by stimulating maturation of DCs to inhibit negative regulation of the Th1 immune response by Tregs. Taken together, our findings suggest that lentinan has prophylactic potential for the treatment of malaria.

The direct effect of IL-12 on tumor cells: IL-12 acts directly on tumor cells to activate NF-kappaB and enhance IFN-gamma-mediated STAT1 phosphorylation.

IL-12 directly acts on T cells and NK cells to induce IFN-gamma production. IFN-gamma plays an important role in anti-tumor effect of IL-12. In spite of various functions of IL-12 on immunocytes, the direct effect of IL-12 on tumor cells has not been fully clarified. The present study investigated the direct effect of IL-12 on eight murine tumor cell lines in vitro. IL-12 did not directly up-regulate expression of MHC class I on tumor cells, but enhanced IFN-gamma-induced up-regulation of MHC class I expression in MC-38, MCA102, MCA205 and MCA207 cells. IL-12 alone did not activate STAT1, but IL-12 enhanced IFN-gamma-mediated STAT1 phosphorylation in MC-38, MCA102, MCA205, MCA207 and Colon-26-NL-17 cells, which expressed IL-12 receptor beta1 mRNA. In the other side, Panc-02, B16-BL6 and 266-6 cells were not affected by IL-12, in which expression of IL-12 receptor beta1 mRNA was not detected. Anti-IL-12 mAb inhibited the direct effect of IL-12 on MC-38 cells. Moreover, nuclear localization of NF-kappaB was observed after stimulation of IL-12 or IL-12 p40 in MC-38 and Colon-26-NL-17 cells, but not in Panc-02 cells. These findings suggest that IL-12 directly acts on tumor cells through IL-12 receptor beta1 to activate NF-kappaB and enhance IFN-gamma-mediated STAT1 phosphorylation.