Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella).

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rß1) and IL-12Rß2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rß1 (gcIl-12rß1), gcIl-12rß2a and gcIl-12rß2b. In silico analysis showed that gcIl-12rß1 and gcIl-12rß2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rß2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rß1 and gcil12rß2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rß2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rß1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rß1 being genuine orthologues of mammalian IL-12Rß1. Moreover, our data showed that gcIl-12rß1 and gcIl-12rß2a but not gcIl-12rß1 and gcIl-12rß2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

Effects of hyperbaric oxygen therapy on the expression levels of the inflammatory factors interleukin-12p40, macrophage inflammatory protein-1ß, platelet-derived growth factor-BB, and interleukin-1 receptor antagonist in keloids.

BACKGROUND: Our study aimed to screen and explore the expression of inflammatory factors in keloid patients and to investigate how hyperbaric oxygen (HBO) therapy affects the expression levels of interleukin-12p40 (IL-12p40), macrophage inflammatory protein-1ß (MIP-1ß), platelet-derived growth factor-BB (PDGF-BB), and interleukin-1 receptor antagonist (IL-1Ra). OBJECTIVE: 30 patients were randomly selected and divided into the following 3 groups: keloid samples from keloid patients treated with HBO therapy (A), keloid samples from keloid patients treated without HBO therapy (B), and normal control skin samples derived from individuals who had no clear scarring (C). Each group included 10 samples. METHODS: Inflammatory factors in the keloid tissues were measured with the MILLIPLEX multiplexed Luminex system. Hematoxylin and eosin staining, immunohistochemical staining, and Western blotting were used to observe the morphological differences in different tissues and the expression levels. RESULTS: The expression levels of inflammatory mediators, including IL-12p40, MIP-1ß, PDGF-BB, and IL-1Ra, in keloid tissues were significantly different from those in samples of normal skin. Hematoxylin and eosin staining showed significantly greater inflammatory infiltration in keloid tissue. Significantly different expression levels were observed in group A, B, and C. CONCLUSION: Significantly altered levels of inflammatory factors in the samples from keloid patients were observed, suggesting that formation of a keloid is potentially related to inflammatory responses. HBO therapy could significantly affect the expression levels of IL-12p40, MIP-1ß, PDGF-BB, and IL-1Ra, indicating that the effects of HBO therapy are associated with the attenuation of inflammatory responses.

Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rß1.

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

Distinctive expression of interleukin-23 receptor subunits on human Th17 and γδ T cells.

The interleukin-23 (IL-23) pathway, T helper 17 (Th17) cells and γδ T cells, which respond to IL-23, have major pro-inflammatory roles. We have used unique IL-23 receptor (IL-23R) subunit-specific monoclonal antibodies, X67 and X68, and IL-12 receptor beta-1 subunit (IL-12Rß1) expression levels to evaluate the IL-23R complex on CD4 αß TCR Th17 cells and on γδ T cells. Both IL-23R and IL-12Rß1 subunits constitute the functional IL-23R. Expression of the IL-23R subunit by cultured Th17 cells was heterogeneous. Th17 cells expressed consistent high levels of the IL-12Rß1 subunit, which appeared a better predictor of responsiveness to IL-23 than the expression of the IL-23R subunit. Moreover, sorting memory CD4 T cells by high IL-12Rß1 expression selectively enriched cells committed to IL-17 production from the blood. IL-23R expression was also observed on freshly isolated and cultured γδ T cells and the cultured γδ T cells were not responsive to IL-23.

Effect of in ovo administration of inulin and Lactococcus lactis on immune-related gene expression in broiler chickens.

OBJECTIVE To evaluate the effect of in ovo administration of inulin and Lactococcus lactis on immune-related gene expression in broiler chickens. ANIMALS 45 Ross broilers. PROCEDURES On day 12 of embryonic development, 360 eggs were equally allocated among 3 treatment groups and injected with 0.2 mL of a solution that contained 1.76 mg of inulin (prebiotic group) or 1.76 mg of inulin enriched with 1,000 CFUs of L lactis subsp lactis 2955 (synbiotic group), or they were injected with 0.2 mL of saline (0.9% NaCl) solution (control). At 1, 14, and 35 days after hatching, 5 male birds from each group were euthanized, and the spleen and cecal tonsils were harvested for determination of interleukin (IL)-4, IL-6, IL-8, IL-12p40, IL-18, cluster of differentiation 80, interferon-ß, and interferon-γ expression by means of a reverse transcription quantitative PCR assay. Gene expressions in the cecal tonsils and spleens of chickens in the prebiotic and synbiotic groups were compared with those of control chickens at each tissue collection time. RESULTS Compared with control birds, immune-related gene expression was downregulated in birds in the prebiotic and synbiotic groups, and the magnitude of that downregulation was more pronounced in the cecal tonsils than in the spleen and increased with age. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that in ovo administration of a prebiotic or synbiotic to broilers was associated with downregulation of immune-related gene expression in the cecal tonsils and spleen. The magnitude of that downregulation increased with age and was most likely caused by stabilization of the gastrointestinal microbiota.

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPß recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

Non-canonical interleukin 23 receptor complex assembly: p40 protein recruits interleukin 12 receptor ß1 via site II and induces p19/interleukin 23 receptor interaction via site III.

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor ß1 (IL-12Rß1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rß1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rß1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rß1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rß1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rß1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rß1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.

Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response.

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rß1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rß1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rß-IL-12Rß2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.

Distinct pathways of humoral and cellular immunity induced with the mucosal administration of a nanoemulsion adjuvant.

Nasal administration of an oil-in-water nanoemulsion (NE) adjuvant W805EC produces potent systemic and mucosal, Th-1- and Th-17-balanced cellular responses. However, its molecular mechanism of action has not been fully characterized and is of particular interest because NE does not contain specific ligands for innate immune receptors. In these studies, we demonstrate that W805EC NE adjuvant activates innate immunity, induces specific gene transcription, and modulates NF-κB activity via TLR2 and TLR4 by a mechanism that appears to be distinct from typical TLR agonists. Nasal immunization with NE-based vaccine showed that the TLR2, TLR4, and MyD88 pathways and IL-12 and IL-12Rß1 expression are not required for an Ab response, but they are essential for the induction of balanced Th-1 polarization and Th-17 cellular immunity. NE adjuvant induces MHC class II, CD80, and CD86 costimulatory molecule expression and dendritic cell maturation. Further, upon immunization with NE, adjuvant mice deficient in the CD86 receptor had normal Ab responses but significantly reduced Th-1 cellular responses, whereas animals deficient in both CD80 and CD86 or lacking CD40 failed to produce either humoral or cellular immunity. Overall, our data show that intranasal administration of Ag with NE induces TLR2 and TLR4 activation along with a MyD88-independent Ab response and a MyD88-dependent Th-1 and Th-17 cell-mediated immune response. These findings suggest that the unique properties of NE adjuvant may offer novel opportunities for understanding previously unrecognized mechanisms of immune activation important for generating effective mucosal and systemic immune responses.

Human interleukin 23 receptor induces cell apoptosis in mammalian cells by intrinsic mitochondrial pathway associated with the down-regulation of RAS/mitogen-activated protein kinase and signal transducers and activators of transcription factor 3 signaling pathways.

The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-ß1 chain. The IL-12R-ß1 heterodimerizes with IL-23R and IL-12R-ß2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-ß2 has been shown to function as a tumor suppressor gene and apoptotic inducer. However, whether IL-23R also functions in cell apoptosis is currently unknown. In this study, we demonstrate for the first time that overexpression of IL-23R markedly induces cell apoptosis in both 293ET and HeLa cells. The activations of caspase 3 and caspase 9 are induced by IL-23R. Mechanistic study reveals that IL-23R markedly inhibits RAS/MAPK and STAT3 but not STAT1 and PI-3K/Akt signaling pathways in both 293ET and HeLa cells. Overexpression of IL-23R significantly up-regulates IL-12Rß1 expression but not IL-23α and IL-12ß expressions in both cell lines. Therefore, our data strongly indicates that IL-23R is able to induce cell apoptosis by activating the intrinsic mitochondrial pathways associated with the inhibition in RAS/MAPK and STAT3 activations in mammalian cells.

Multifocal Tuberculous osteomyelitis: possible inherited interferon gamma axis defect.

Multifocal Tuberculous (TB) osteomyelitis is an extremely rare entity in immunocompetent individuals. The authors report a 19-mo-old girl with multifocal TB osteomyelitis which resolved completely following institution of four drug antituberculous treatment for 1 y and detailed immunological evaluation was found to be normal but as the response of interferon gamma (IFN γ) in terms of production of IL12p40 was severely impaired, she is suggested to have a possible defect in the interferon gamma axis even though the exact defect in the pathway could not be delineated. This case report reiterates the need for detailed immunological evaluation in children presenting with multifocal TB osteomyelitis.

Disseminated Mycobacterium genavense infection in a patient with a novel partial interleukin-12/23 receptor ß1 deficiency.

A patient presented with late onset disseminated nontuberculous mycobacterium (NTM) infection due to a novel interleukin-12/interleukin-23 receptor ß1 (IL-12/IL-23Rß1) mutation, r.1561C>G, leading to the amino acid substitution R521G. This is the second patient reported with a partial IL-12/IL-23Rß1 defect.

The role of interleukin-12 on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis.

Myeloid-derived suppressor cells (MDSC) are important to the tumour microenvironment as they actively suppress the immune system and promote tumour progression and metastasis. These cells block T-cell activation in the tumour microenvironment, preventing anti-tumour immune activity. The ability of a treatment to alter the suppressive function of these cells and promote an immune response is essential to enhancing overall therapeutic efficacy. Interleukin-12 (IL-12) has the potential not only to promote anti-tumour immune responses but also to block the activity of cells capable of immune suppression. This paper identifies a novel role for IL-12 as a modulator of MDSC activity, with implications for IL-12 as a therapeutic agent. Treatment with IL-12 was found to alter the suppressive function of MDSC by fundamentally altering the cells. Interleukin-12-treated MDSC exhibited up-regulation of surface markers indicative of mature cells as well as decreases in nitric oxide synthase and interferon-γ mRNA both in vitro and in vivo. Treatment with IL-12 was also found to have significant therapeutic benefit by decreasing the percentage of MDSC in the tumour microenvironment and increasing the percentage of active CD8(+) T cells. Treatment with IL-12 resulted in an increase in overall survival accompanied by a reduction in metastasis. The findings in this paper identify IL-12 as a modulator of immune suppression with significant potential as a therapeutic agent for metastatic breast cancer.

Influenza virus vaccination induces interleukin-12/23 receptor beta 1 (IL-12/23R beta 1)-independent production of gamma interferon (IFN-gamma) and humoral immunity in patients with genetic deficiencies in IL-12/23R beta 1 or IFN-gamma receptor I.

To investigate whether protective immune responses can be induced in the absence of normal interleukin-12/23/gamma interferon (IL-12/23/IFN-gamma) axis signaling, we vaccinated with the seasonal influenza virus subunit vaccine two patients with complete IL-12/23 receptor beta1 (IL-12/23R beta 1) deficiencies, two patients with partial IFN-gamma receptor I (pIFN-gamma RI) deficiencies, and five healthy controls. Blood samples were analyzed before, 7 days after, and 28 days after vaccination. In most cases, antibody titers reached protective levels. Moreover, although T-cell responses in patients were lower than those observed in controls, significant influenza virus-specific T-cell proliferation, IFN-gamma production, and numbers of IFN-gamma-producing cells were found in all patients 7 days after the vaccination. Interestingly, influenza virus-specific IFN-gamma responses were IL-12/23 independent, in striking contrast to mycobacterium-induced IFN-gamma production. In conclusion, influenza virus vaccination induces IL-12/23-independent IFN-gamma production by T cells and can result in sufficient humoral protection in both IL-12/23R beta 1- and pIFN-gamma RI-deficient individuals.

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275.

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.

Increased IL-23p19 expression in multiple sclerosis lesions and its induction in microglia.

IL-12 has long been considered important in the pathogenesis of multiple sclerosis. However, evidence from recent studies strongly supports the critical role of IL-12-related proinflammatory cytokine IL-23, but not IL-12, in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of this disease. The role of IL-23 in the CNS immunity of multiple sclerosis patients has not been elucidated; nor is it known whether human microglia produce this cytokine. In this study we investigated the expression of IL-23p19 and p40, with its key subunit p19 as the focus, in histologically characterized CNS specimens from multiple sclerosis and control cases using in situ hybridization and immunohistochemistry. A significant increase in mRNA expression and protein production of both subunits of IL-23 was found in lesion tissues compared with non-lesion tissues. Double staining showed that activated macrophages/microglia were an important source of IL-23p19 in active and chronic active multiple sclerosis lesions. We also detected IL-23p19 expression in mature dendritic cells which were preferentially located in the perivascular cuff of active lesions. The finding that human microglia produce IL-23 was further confirmed by the inducible production of IL-23p19 and p40 in cultured human microglia in vitro upon different Toll-like receptor stimulations. Taken together, these findings on the expression of IL-23p19 in multiple sclerosis lesions may lead to a better understanding of the events culminating in human multiple sclerosis.