IL-12p40 Expression Research Articles

SCIMP is a universal Toll-like receptor adaptor in macrophages.

In innate immune cells, pathogens and danger signals activate TLRs, unleashing potent and tailored inflammatory responses. Previously, we reported that an immune-specific transmembrane adaptor, SLP adaptor and CSK interacting membrane protein (SCIMP), interacts with TLR4 via direct binding to its cytoplasmic TIR domain. SCIMP scaffolds a Src family kinase, Lyn, for TLR4 phosphorylation and activation. Consequently, SCIMP is able to direct selective production of the proinflammatory cytokines IL-6 and IL-12p40 downstream of TLR4 in macrophages. Here, we set out to investigate whether SCIMP also acts as an adaptor for other TLR family members. We report here that SCIMP is phosphorylated and activated in response to agonists of multiple TLRs, including TLR2, TLR3, TLR4, and TLR9. SCIMP also interacts with TLRs that are known to signal from both the cell surface and endosomal compartments. In so doing, this transmembrane adaptor presents Lyn, along with other effectors such as Grb2, Csk, and SLP65, to multiple TLRs during cellular activation. CRISPR-mediated knockout or silencing of SCIMP in macrophages alters TLR signaling outputs and the production of IL-6 and IL-12p40 downstream of multiple TLRs, and upon challenge with live bacteria. Furthermore, the selectivity in cytokine responses is preserved downstream of TLR3, with inducible expression of Il-12p40 and IL-6, but not IFNβ, being SCIMP dependent. SCIMP is thus a universal TLR adaptor for scaffolding the Lyn tyrosine kinase and its effectors to enable responses against a wide range of danger signals.

Abstract 2346: Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells

Abstract PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), a prominent mutagen/carcinogen is a heterocyclic amine found in meat cooked at high temperature and in tobacco. PhIP is known to induce liver, colon, skin, prostate, and breast cancer as evident from several in vivo and in vitro studies. Our previous studies have demonstrated that curcumin significantly inhibits PhIP-induced DNA adduct formation and DNA double-strand breaks with the concomitant decrease in reactive oxygen species (ROS) production via multiple molecular targets. We hypothesize that PhIP induced ROS production induces inflammatory proteins and co-treatment with curcumin would suppress the induced inflammation. Therefore, human breast epithelial cells (MCF 10A) were used to test the expression of inflammatory proteins. MCF 10A cells were treated with PhIP alone and in presence of curcumin. Untreated cells and treated only with curcumin served as control. Human Inflammation array (Ray Biotech) was used to analyze the expression of forty inflammatory proteins. Results show that the expression of twenty inflammatory proteins is induced in MCF 10 A cells when treated with PhIP as compared to the controls. Presence of curcumin reduces the induced expression of these twenty inflammatory proteins. However, three inflammatory proteins (IL-8, IL 12p40 and ICAM-1) showed a significantly higher level of expression with PhIP and curcumin co-treated cells as compared to cells treated with PhIP alone. It is shown that higher expression of IL-8, IL 12p-40 and ICAM-1 is involved in chemotactic factor involve in attracting the neutrophils, basophils; activation of T and NK cells which might be helping in coordinating the inflammation process. Hence the present study suggests that curcumin is a promising natural compound to revert the effect of PhIP induced inflammation. Citation Format: Ashok Jain. Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2346.

Abstract 102: Ferrichrome suppresses pancreatic tumor growth via targeting TAMs and enhancing CD8+ T-cells infiltration

Abstract Background: Pancreatic cancer (PanCa) is one of the most lethal malignancy with a very poor survival rate in patients due to inadequate treatment options. Accumulating evidences suggest that tumor associated macrophages (TAMs) and reduce infiltration of CD8+ T-cell population provide tumor suppressive environment leading to advanced growth and metastasis of PanCa. Thus, targeting TAMs and enhancing infiltration of CD8+ T-cells to the tumor site by non-toxic agents could be an effective therapeutic approach for the management of PanCa. In this study, we demonstrate that a novel probiotic-derived agent (ferrichrome) inhibits pancreatic tumor growth in syngeneic mouse model via modulating TAMs and increasing infiltration of CD8+ T-cells. Methods: RAW264.7 cells, murine peritoneal macrophages, mouse pancreatic cancer cells (UN-KC-6141) were used for this study. Effect of ferrichrome on the expression of pro (IL-12, p40, iNOS, IL-6), anti-inflammatory cytokines (MRC1 and Arginase-1) and iron metabolism markers (ferritin and ferroportin) in macrophages and co-culture systems was analyzed by qPCR. Effect of ferrichrome on metastatic phenotypes of PanCa cells was analyzed in a co-culture model of RAW264.7 and UN-KC-6141 cells. Therapeutic efficacy of ferrichrome was determined in syngeneic mouse model. Tumor immune infiltrating cells were analyzed in excised tumors by FACS, double immunofluorescence and immunohistochemistry analyses. Results: Ferrichrome treatment reversed the polarization of M2 macrophages towards the M1 phenotype as observed by increase expression of IL-12p40, iNOS, IL-6 and decrease expression of MRC1 and Arginase-1. Ferrichrome significantly (P<0.01) reduced the RAW264.7 cells induced migration and invasive potential of UN-KC-6141 cells. We further investigated the molecular mechanisms of ferrichrome induced M1 macrophage phenotype. Our results revealed that ferrichrome-induced M1 polarization of macrophages via upregulation of iron-sequestration protein (ferritin) and downregulation of iron-export protein (ferroportin). Ferrichrome administration (50 µg) intratumorally for 5 days per week for 26 days significantly (P<0.01) inhibited growth of xenograft tumors. We observed an increase of CD8+ T-cell and iNOS+F4/80+ macrophages infiltration and a decrease in the expression of CD163+ macrophages in resected tumors of ferrichrome treated mice. Conclusion: Our results strongly suggest that ferrichrome inhibits the tumor growth of PanCa via targeting TAMs and enhancing CD8+ T-cell infiltration. Ferrichrome could be used alone or in combination with current therapeutic regimens for the treatment of advanced PanCa. Citation Format: Mehdi Chaib, Bilal Bin Hafeez, Sonam Kumari, Mohammed Sikander, Hassan Mandil, Liza Makowski, Ajeeth Kumar Pingili, Elham Hatami, Advait Shetty, Dan Nirnoy, Murali Mohan Yallapu, Meena Jaggi, Subhash Chauhan. Ferrichrome suppresses pancreatic tumor growth via targeting TAMs and enhancing CD8+ T-cells infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 102.

Abstract 3195: Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity

Abstract Preclinical and clinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid) delivered intratumorally via electroporation (TAVO) induces local expression of IL-12p70, converting immunologically excluded tumors into inflamed immunogenic lesions, which is fundamental to generating objective responses in both treated and untreated distant tumors. Recent optimization of electroporation parameters and plasmid design has yielded a significant increase in intratumoral expression and anti-tumor efficacy in preclinical models. To further develop this platform, longitudinal biomarker data from TAVO clinical trials was interrogated to identify key immunological components associated with an effective therapy. This analysis revealed that the composition of TAVO-inflamed lesions, specifically the frequency of CD8+ TIL and the chemokines necessary for their maintenance relative to suppressive intratumoral immune subsets, coincided with clinical responses. We therefore developed a plasmid that encodes both IL-12 and the effector T cell chemokine, CXCL9, which, when transfected into a tumor, are functionally active both in vitro as well as in vivo, yielding abscopal responses and increased survival. To further amplify this technology, a strategy was developed to engage the highly relevant and recently described bystander non-tumor reactive T cells in the tumor microenvironment (TME). To accomplish this pan-T cell amplification, a separate therapeutic plasmid encoding membrane-bound anti-CD3 scFv (145-2C11) was used to decorate transfected tumors with the potent polyclonal T cell stimulator. Membrane expression of anti-CD3 in vivo was confirmed by immunoblot and flow cytometry. Combination of IL-12/CXCL9 with anti-CD3 delivered by electroporation (collectively, “SPARK”) increased both antigen-specific and polyclonal T cells responses, demonstrated by in vivo CTL and proliferation assays. Finally, SPARK upregulated the transcription of key immunological genes, leading to regression of both treated and untreated tumors, which heightens its ability to reshape the TME and drive systemic anti-tumor immunity. In conclusion, this data supports a model whereby IL-12 in concert with CXCL9 inflames the lesion, leading to a brisk T cell infiltrate, which in sequence with anti-CD3 stimulation, drives a broad yet robust systemic T cell response. SPARK represents a significant advancement in cytokine-based immunotherapy. Note: This abstract was not presented at the meeting. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Kurt Sakurada, Jack Lee, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3195.

Abstract 5017: MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment

Abstract Patients who respond to PD-L1 / PD-1 immune checkpoint blockade tend to have an inflamed, TH1 polarised tumor microenvironment (TME), characterised by expression of interferon-γ (IFNγ) and PD-L1. Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor immunity. As a central mediator of TH1 immune responses, interleukin 12 (IL-12) directly induces IFNγ release from activated NK, NKT and T cells, and is known to play a key role in driving anti-tumor responses. However systemic recombinant IL-12 was poorly tolerated in early clinical trials. We therefore designed MEDI1191 as a novel IL-12-based therapy designed for injection directly into tumors, composed of a lipid nanoparticle (LNP)-formulated mRNA encoding human IL-12. We previously reported that intratumoral (IT) mouse (m) IL-12 mRNA, the surrogate for MEDI1191, promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models. Here, we demonstrate that IFNγ is also required for the anti-tumor activity of mIL-12 mRNA. A single dose of mIL-12 mRNA significantly increased expression of IFNγ and TH1 genes in MC38 tumor-bearing mice. Treatment with an IFNγ neutralising antibody blocked mIL-12 mRNA anti-tumor activity in this model. In addition, we report here that MC38 tumor rejection in response mIL-12 mRNA / anti-PD-L1 combination therapy correlates with increased cytotoxic T cell infiltration into tumors, and expansion of tumor-reactive T cells in the periphery. We next investigated the pharmocodynamic activity of MEDI1191 in patient tumor-derived models. A single IT dose of MEDI1191 induced human IL-12p70 expression in mice bearing four different patient-derived xenograft tumors. Furthermore, in an ex vivo patient tumor slice culture assay, MEDI1191 induced dose-dependent IL-12 release, IFNγ expression and upregulation of TH1-signature gene expression. IL-12 protein secretion was induced in slices of all patient tumors tested. However, the magnitude of the IFNγ response to MEDI1191 varied between patient tumors. Quantification of the tumoral T cell and NK cell numbers within the patient tumor samples revealed a positive correlation between MEDI1191-induced IFNγ release and baseline tumor NK infiltrate. These preclinical data demonstrate the potential for MEDI1191 to induce IFNγ-dependent TH1 transformation of the TME, and support the development of MEDI1191 as a potential treatment for patients with solid tumors, alone and in combination with inhibitors of the PD-1/PD-L1 T cell checkpoint. Citation Format: Nadia Luheshi, Susannah Hewitt, Fabien Garcon, Shannon Burke, Amanda Watkins, Kristen Arnold, John Zielinski, Philip Martin, Michael Sulikowski, Christopher Bagnall, Jean-Martin Lapointe, Gordon Moody, Han Si, Christopher Morehouse, Robert W. Wilkinson, Ronald Herbst, Joshua Frederick. MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5017.

Local Cytokine Expression Profiling in Patients with Specific Autoimmune Uveitic Entities

ABSTRACTPurpose: To evaluate expression of cytokines GM-CSF, IL-11, IL-12p40, IL-12p70, IL-27p28, IL-35, APRIL, BAFF, TWEAK, and LIGHT in uveitis.Methods: Aqueous humor samples from patients with active uveitis associated with Behçet’s disease (BD), sarcoidosis, HLA-B27-related inflammation, and Vogt-Koyanagi-Harada (VKH) disease and control patients were assayed with a multiplex assay.Results: Comparing all patients to controls, GM-CSF, IL-11, IL-12p40, APRIL, and BAFF were significantly increased, whereas LIGHT was significantly decreased. IL-11 and BAFF were the most strongly upregulated, being elevated 19.7-fold and 14.1-fold, respectively, compared with controls. IL-11 was significantly highest in HLA-B27 uveitis. GM-CSF, IL-11, and IL-12p40 were significantly higher in nongranulomatous uveitis (BD and HLA-B27) than in granulomatous uveitis (sarcoidosis and VKH), whereas APRIL and TWEAK were significantly higher in granulomatous uveitis.Conclusions: IL-11-driven immune responses might be more potent in nongranulomatous uveitis, particularly in HLA-B27 uveitis. BAFF and APRIL might contribute to B cell-driven autoimmune response in uveitis.

CNS-Specific Synthesis of Interleukin 23 Induces a Progressive Cerebellar Ataxia and the Accumulation of Both T and B Cells in the Brain: Characterization of a Novel Transgenic Mouse Model.

Interleukin 23 (IL-23) is a key mediator in neuroinflammation in numerous autoimmune diseases including multiple sclerosis (MS). However, the pathophysiology of IL-23 and how it contributes to neuroinflammation is poorly defined. To further clarify the role of IL-23 in CNS inflammation, we generated a transgenic mouse model (GF-IL23) with astrocyte-targeted expression of both IL-23 subunits, IL-23p19, and IL-23p40. These GF-IL23 mice spontaneously develop a progressive ataxic phenotype, which corresponds to cerebellar tissue destruction, and inflammatory infiltrates most prominent in the subarachnoidal and perivascular space. The CNS-cytokine milieu was characterized by numerous inflammatory mediators such as IL-17a and IFNγ. However, the leukocytic infiltrates were surprisingly predominated by B cells. To further examine the impact of the CNS-specific IL-23 synthesis on an additional systemic inflammatory stimulus, we applied the LPS-induced endotoxemia model. Administration of LPS in GF-IL23 mice resulted in early and pronounced microglial activation, enhanced cytokine production and, in sharp contrast to control animals, in the formation of lymphocytic infiltrates. Our model confirms a critical role for IL-23 in the induction of inflammation in the CNS, in particular facilitating the accumulation of lymphocytes in and around the brain. Thereby, CNS-specific synthesis of IL-23 is able to induce a cascade of inflammatory cytokines leading to microglia activation, astrocytosis, and ultimately tissue damage. The presented transgenic model will be a useful tool to further dissect the role of IL-23 in neuroinflammation.

Outcomes of ERK Signaling Differ in Macrophages and Dendritic Cells

Abstract Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety of immune responses. Pathogen-induced cell signaling and associated outcome research is abundant in macrophages, but minimal in dendritic cells. We investigated the role of ERK signaling in the induction of cytokines such as IL-12p40 and IL-10 following activation by Pam3Cys or Mycobacterium tuberculosis (Mtb) H37Ra. ERK ablation was accomplished pharmacologically with MERK1/2 inhibitor U0126 or genetically by using cells from Tpl2−/− mice (TPL2 connects TLR to ERK). We utilized western blotting to examine ERK activation, ELISA to assess cytokine production, and qRT-PCR to investigate mRNA levels. In macrophages, blockade of ERK signaling inhibited IL-10 production and increased IL-12p40 production (6–10 fold increase in IL-12p40 mRNA and 3–4 fold increase in protein). In dendritic cells, ERK blockade similarly inhibited IL-10 production, but produced a very different change in IL-12p40 expression: ERK blockade decreased IL-12p40 production (2-fold decrease in IL-12p40 mRNA and protein). This result suggests that the impact of ERK signaling in response to these stimuli differs between macrophages and dendritic cells for a subset of ERK-regulated genes. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Future studies will examine other genes and proteins involved in infection responses. Greater understanding of the role that ERK signaling plays in different cell types may inform the development of host-directed therapy for infections such as tuberculosis.

Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

Expression profiling of cytokine-related genes in Brucella abortus infected cattle

ABSTRACT The present investigation was undertaken to assess the rhythmic change in the expression profile of cytokine-related genes in the PBMC of Brucella abortus infected cattle. The whole blood samples were collected from cattle from the state of Rajasthan in Western India. All 20 samples of PBMC cultured cells from the 10 diseased cattle serologically positive for brucellosis, and 10 serologically negative healthy controls, were harvested, and the expression of cytokine-related genes was normalized relative to β-actin for their quantification by SYBR green-based real-time PCR. The subsequent analysis was based on the comparisons within treatment group (unstimulated or B. abortus stimulated) and between infection groups (healthy control versus diseased). The expression profiling of cytokine genes revealed differential expression of IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-12p40 and iNOS by PBMCs from healthy cows and Brucella positive cows even without stimulation with Brucella abortus antigen. This unique expression profile with enhanced expression of genes encoding IFN-γ, IL-1β, IL-6 and iNOS, and reduced expression of TNF-α, IL-4 and IL-12p40; indicates changing of the dynamic immune responses from a predominantly Th1-like pattern to a Th2-like pattern with the development of a chronic form of bovine brucellosis. The rhythmic change in immunological profiles conclusively analyzed in the present investigation will help to implement control measures in Western India. Abbreviations: PBMC: peripheral blood mononuclear cells; PCR: Polymerase chain reaction; IFN: Interferon; TNF: Tumour necrosis factor; IL: Interleukin; iNOS: Inducible nitric oxide synthase; Th: T helper

Aberrant methylation of the IL-12B promotor region contributes to the risk of developing ovarian endometriosis.

Studies have shown that aberrant expression of IL-12p40, which is encoded by the interleukin-12B (IL-12B) gene, may be involved in the development of endometriosis. In this study, we investigated the role of aberrant methylation of the IL-12B promoter region and its associated expression in the development of ovarian endometriosis. By using pyrosequencing, we analyzed the methylation level of the IL-12B promoter region in eutopic and ectopic endometrium of patients with ovarian endometriosis and normal endometrium of control women. The expression of IL-12B mRNA was detected by quantitative real-time PCR. The results showed that the methylation level of the IL-12B promoter region in ectopic and eutopic endometrium of patients with ovarian endometriosis was significantly lower than that in endometrium of women without endometriosis ( p < 0.001 and p = 0.041, respectively). In contrast, mRNA levels were significantly increased in ectopic and eutopic endometrium of patients with ovarian endometriosis compared to those in endometrium of women without endometriosis ( p < 0.001 and p = 0.042, respectively). Correlation analysis showed that the methylation level of the IL-12B promoter region was negatively correlated with mRNA levels of IL-12B ( p < 0.001). Our data suggested that aberrant methylation of the IL-12B promoter region may be responsible for aberrant IL-12B mRNA expression in endometrium tissue of women, which may be associated with the development of ovarian endometriosis in northern Chinese women.

Th17 responses to autologous dendritic cell vaccine.

132 Background: Dendritic cells (DC) loaded with autologous tumor antigens in vitro, can induce a Th17 phenotype preceding a Th1 type response. We examined this in patients with metastatic melanoma who had been treated in a randomized trial with either DC loaded with autologous tumor antigens (ATA) from self-renewing tumor cells (DCV) or irradiated tumor cells (TCV). Methods: Blood samples were obtained at baseline, one week before three weekly vaccinations (week-0), and at week-4, (one week after the third vaccination). They were analyzed for growth factors, tumor markers and biomarkers using a quantitative, multiplex ELISA. Monocytes, lymphocytes, loaded and unloaded DC were analyzed for cytokine production and intercellular responses using mixed lymphocyte co-cultures (MLC). Results: When compared to baseline or the TCV arm, DCV-treated patients showed reductions in tumor markers, inflammation and angiogenesis in parallel with Th17 pathway activation, IL12 p70 heterodimer expression was detected in monocytes and DC only after antigen loading. The IL12 p40 fraction was present after differentiation and was enhanced after antigen loading. Similarly, the p19 component of IL23 was expressed before DC loading and later enhanced. MLC showed induction of Th17, Th1 and CD8 subpopulations. In the presence of antigen-loaded DCs, the FOXp3 cells were reduced in number, an effect that was canceled by neutralizing the IL12p40 subunit or by the adding tumor cells alone in the MLC. Conclusions: DC produced ex vivo can activate multiple cytotoxic pathways depending on IL12 subunits. Expression of IL23p19 and the shared IL12p40 fraction are responsible for Th1 priming (IL12 incomplete) and Th17 induction (IL23 complete), determinants of the reactive phenotype of naïve DCs. IL12 p70 heterodimer expression after DC loading enables a targeted Th1 type response while neutralizing the IL12 subunit p40, enhances Foxp3 positive T-regs. DC vaccines produced ex vivo could induce a Th1/Th17 cytotoxic response against tumors. In the absence of antigens, however, The Th17 phenotype can become tolerizing in the microenvironment. The findings are important for applications in settings in which tumor infiltration with Th17 correlates with survival, such as in advanced ovarian cancer. Clinical trial information: NCT00436930.

P2Y6 Signaling Controls an Innate Alveolar Macrophage-NK Cell Axis That Dampens Type 2 Lung Immunopathology

Type 6 purinergic (P2Y6) receptors are high affinity G protein-coupled receptors (GPCRs) for uridine diphosphate (UDP). We found that P2Y6 receptor expression during the sensitization phase plays a critical role in preventing sensitization to the allergen. In this study, we sought to identify key steps and cell types response for the protective effect of P2Y6 receptors. p2ry6(flox/flox);rosa26creER/+ and p2ry6(flox/flox);+/+ controls were treated with tamoxifen starting either 10 days before the initial sensitization. The mice were sensitized with an extract from Dermatophagoides farinae (Df) intranasally on days 0 and 1. We examined BAL fluid from P2ry6flox/flox/Cre/+ mice and +/+ controls for Th1 cytokines by alveolar macrophages, as well as the NK cell activation, during the sensitization phase. BAL fluid levels of IL-12p40, expression of IFNγ, and M1-cytokine/chemokine transcripts by BAL fluid cells increased sharply in response to 2 sensitizing doses of Df in +/+ but not in P2ry6flox/flox/Cre/+ mice. Alveolar macrophages were the dominant source of P2Y6 receptor-dependent IL-12p40 expression, driving IFNγ production by NK cells. The IL-12-restored P2ry6flox/flox/Cre/+ mice showed significantly increased expression of IFNγ at day2 and decreased BAL fluid eosinophils at day 16, compared to the Df-treated +/+ controls. Antibody depletion of NK cells before sensitization abolished the protective effect of P2Y6 receptors. Alveolar macrophages use UDP-P2Y6 signalling to drive an innate IL-12/NK cell axis that strongly influences the outcome of respiratory allergen exposure.

Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity

SummaryProduction of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4+ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer’s patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c+ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c+ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.

Assessment of intralesional cytokine profile of cutaneous leishmaniasis caused by Leishmania donovani in Sri Lanka

BackgroundCytokines play a vital role in the host immune response to infection by initiating the healing process and/or accelerating the progression of the disease in cutaneous leishmaniasis (CL). Very little evidence is available on cytokine profiles and their regulatory function in CL patients in Sri Lanka. The aim of this study was to determine the cytokine expression pattern of IFN-γ, IL-4, IL-11 and IL-12p40 in CL patients and in healthy volunteers. Patients with suspected CL lesions attending to the Dermatology Clinic at the Anuradhapura Teaching Hospital were included in the study. Reverse transcription real time polymerase chain reaction (real-time RT-PCR) was performed to determine the relative expression level of target cytokines. Expression levels were quantified by 2- ∆∆CT equation.ResultsThe expression of cytokines IFN-γ, IL-4, IL-11 and IL-12p40 were significantly higher in CL patients compared to healthy volunteers (p < 0.05). There was a significant association between the expression of IFN-γ and the duration of the lesion (p = 0.021). Wet CL lesions showed significantly higher expression of IL-4, IL-11 and IL-12p40 (p = 0.039, 0.018 and 0.021 respectively) compared to dry lesions. Papulo-nodular lesions showed significantly high expression of IFN-γ (p = 0.023). However, cytokine expression was not significantly associated with the number, size and the locations of lesions.ConclusionsThe expression levels of all cytokines tested in the present study were significantly (p < 0.05) high in CL patients. Th1 response (IFN-γ and IL-12p40) had higher expression levels compared to Th2 (IL-4) and IL-11 in CL patients.

Interleukin 12B mRNA level and rs3212227 genotyping in peripheral blood mononuclear cells of inflammatory bowel disease patients

Inflammatory bowel disease (IBD) is a multifactorial disorder. Single nucleotide polymorphisms (SNPs) in the IL-12 gene, which are the main factors to regulate the immune reaction, play an important role in the production of IL-12 molecules. The aim of this study was to evaluate the correlation between the SNP on position +1188 of the 3 ′ UTR region of the IL-12 p40 subunit gene and expression of the IL-12 p40 gene. This case-control study was performed with 102 patients with IBD and 107 healthy people. PCR-RFLP and comparative real-time PCR were performed to assess the association between genotype and IL-12 gene expression. The frequency of AA, CA, and CC genotypes of this gene at position +1188 was calculated to be 58.8%, 32.4%, and 8.8% in patients and 61.7%, 26.2%, and 12.1% in the control group, respectively, with no significant difference between the two groups (IL- 12B rs3212227: AA (Reference 1), CA (P = 0.407); OR (95% CI) 0.771 (0.418–1.424), CC (P = 0.561); OR (95% CI) 1.313 (0.524–3.292)). Also, the IL-12B mRNA expression level was compared between IBD patients and healthy controls and demonstrated a significant association (R 2 0.136, 95% CI 1.892–3.872, P < 0.0001). Our results show that IL-12B expression in IBD may be associated with altered immune and inflammatory responses.

Interleukin-12p35 knockout promotes macrophage differentiation, aggravates vascular dysfunction, and elevates blood pressure in angiotensin II-infused mice

Numerous studies have demonstrated that inflammation is involved in the progression of hypertension. Inflammatory cytokines interleukin (IL)-12 and IL-35 belong to the IL-12 cytokine family and share the same IL-12p35 subunit. Accumulating evidence has demonstrated that IL-12p35 knockout (IL-12p35 KO) leads to cardiovascular disease by regulating the inflammatory response. This study aimed to investigate whether IL-12p35 KO elevates blood pressure in a hypertension mouse model. Mice with angiotensin (Ang) II infusion showed marked aortic IL-12p35 expression; thus, aortic macrophages may be the main source of IL-12p35. Wild-type and IL-12p35 KO mice were infused with Ang II or saline. IL-12p35 KO promoted M1 macrophage differentiation, amplified the inflammatory response, aggravated vascular dysfunction, and elevated blood pressure in Ang II-treated mice. Then, some Ang II-infused mice were given phosphate buffer saline, mouse recombinant IL-12 (rIL-12), or rIL-35, and the results showed that rIL-12 but not rIL-35 treatment had an antihypertensive effect on Ang II-infused mice. In addition, detection of human plasma IL-12 levels in hypertensive patients and control subjects showed that IL-12 was significantly increased in hypertensive patients when compared with control subjects. In hypertensive patients, IL-12 levels were positively correlated with blood pressure. IL-12p35 KO amplifies the inflammatory response and promotes blood pressure elevation in Ang II-treated mice. In addition, IL-12, but not IL-35, plays a protective role in the Ang II-induced hypertension model. Thus, IL-12 may be a novel therapeutic agent for the prevention and treatment of clinical hypertension.

Effects of Artemisia absinthium L. extracts on the maturation and function of dendritic cells

Objective To investigate the effects of Artemisia absinthium L. (A.absinthium L. ) extracts on the maturation and function of dendritic cells (DCs). Methods A. absinthium water (AAW) and ethanol (AAE) extracts were prepared. DCs were separated into several groups and treated with different concentrations of AAW (containing 5, 50 and 150 μg/ml of polysaccharide) and AAE (containing 0.6, 3 and 6 μg/ml of flavonoid), respectively. Cell viability, antigen phagocytosis and maturation of DCs were detected by flow cytometry. Levels of cytokines were analyzed by ELISA. Western blot assay was performed to analyze the activation of key molecules in NF-κB, mitogen-activated protein kinase (MAPK) and janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. Results AAW promoted the maturation of DCs, significantly decreased antigen phagocytosis and increased cytokine production (IL-12p40 and TNF-α). AAE significantly enhanced the expression of co-stimulatory molecules on the surface of DCs and decreased antigen phagocytosis, but had no significant effect on cytokine production. Moreover, AAE significantly inhibited the LPS-induced expression of TNF-α, IL-12p40 and IL-6. Further analysis revealed that AAW and AAE could activate the phosphorylation of p38, extra-cellular signal regulated kinase (ERK), IKKα/β, NF-κBp65 and JAK2. Besides, AAE could activate the phosphorylation of c-Jun N-terminal kinase (JNK) and inhibit the LPS-induced phosphorylation of inhibitor of NF-κB (IκB-α), IKKα/β, NF-κBp65, p38, ERK and JAK2. Conclusion AAW could enhance immunity and AAE could inhibit inflammation. Key words: Artemisia absinthium L.; Dendritic cell; Cytokine; Antigen phagocytosis

IL-27 amplifies cytokine responses to Gram-negative bacterial products and Salmonella typhimurium infection

Cytokine responses from monocytes and macrophages exposed to bacteria are of particular importance in innate immunity. Focusing on the impact of the immunoregulatory cytokine interleukin (IL)-27 on control of innate immune system responses, we examined human immune responses to bacterial products and bacterial infection by E. coli and S. typhimurium. Since the effect of IL-27 treatment in human myeloid cells infected with bacteria is understudied, we treated human monocytes and macrophages with IL-27 and either LPS, flagellin, or bacteria, to investigate the effect on inflammatory signaling and cytokine responses. We determined that simultaneous stimulation with IL-27 and LPS derived from E. coli or S. typhimurium resulted in enhanced IL-12p40, TNF-α, and IL-6 expression compared to that by LPS alone. To elucidate if IL-27 manipulated the cellular response to infection with bacteria, we infected IL-27 treated human macrophages with S. typhimurium. While IL-27 did not affect susceptibility to S. typhimurium infection or S. typhimurium-induced cell death, IL-27 significantly enhanced proinflammatory cytokine production in infected cells. Taken together, we highlight a role for IL-27 in modulating innate immune responses to bacterial infection.

Interleukin-12p35 Knock Out Aggravates Doxorubicin-Induced Cardiac Injury and Dysfunction by Aggravating the Inflammatory Response, Oxidative Stress, Apoptosis and Autophagy in Mice.

BackgroundRecent evidence has demonstrated that interleukin 12p35 knockout (IL-12p35 KO) is involved in cardiac diseases by regulating the inflammatory response. The involvement of inflammatory cells has also been observed in doxorubicin (DOX)-induced cardiac injury. This study aimed to investigate whether IL-12p35 KO affects DOX-induced cardiac injury and the underlying mechanisms.MethodsFirst, the effect of DOX treatment on cardiac IL-12p35 expression was assessed. In addition, to investigate the effect of IL-12p35 KO on DOX-induced cardiac injury, IL-12p35 KO mice were treated with DOX. Because IL-12p35 is the mutual subunit of IL-12 and IL-35, to determine the cytokine that mediates the effect of IL-12p35 KO on DOX-induced cardiac injury, mice were given phosphate-buffered saline (PBS), mouse recombinant IL-12 (rIL-12) or rIL-35 before treatment with DOX.ResultsDOX treatment significantly increased the level of cardiac IL-12p35 expression. In addition, IL-12p35 KO mice exhibited higher serum and heart lactate dehydrogenase levels, higher serum and heart creatine kinase myocardial bound levels, and greater cardiac dysfunction than DOX-treated mice. Furthermore, IL-12p35 KO further increased M1 macrophage and decreased M2 macrophage differentiation, aggravated the imbalance of oxidants and antioxidants, and further activated the mitochondrial apoptotic pathway and endoplasmic reticulum stress autophagy pathway. Both rIL-12 and rIL-35 protected against DOX-induced cardiac injury by alleviating the inflammatory response, oxidative stress, apoptosis and autophagy.ConclusionsIL-12p35 KO aggravated DOX-induced cardiac injury by amplifying the levels of inflammation, oxidative stress, apoptosis and autophagy. (234 words).